surfaces to inactivate pathogenic microrganisms
TRANSCRIPT
EFFICACY OF PHOTOCATALYTIC NANOCOATINGS ON FOOD CONTACT
SURFACES TO INACTIVATE PATHOGENIC MICROORGANISMS
by
VEERACHANDRA K. YEMMIREDDY
(Under the Direction of Yen-Con Hung)
ABSTRACT
TiO2 is a promising photocatalyst for use in food processing environments as an
antimicrobial coating. The overall goal of this research was to develop physically stable
TiO2 nanocoatings with strong bactericidal property on food contact surfaces. A testing
protocol was developed to determine the photocatalytic bactericidal activity of TiO2
nanoparticles (NPs) in suspension. Among the tested TiO2 NPs, Aeroxide®
P 25 was
found to be the most efficient and achieved a 5 log reduction of bacteria in 3h. Type and
source of TiO2, bacterial cell harvesting conditions, volume of suspension, and intensity
of UV-A light had significant effect on the log reduction. Further, the effect of food
organic matter on bactericidal property of TiO2 NPs was investigated. Increasing the
concentration of organic matter decreased the bactericidal efficacy of TiO2. A linear
correlation was observed between chemical oxygen demand (COD) and total phenolics as
well as COD and protein contents. An empirical equation in the form of “Y=me-nX
”
(where Y is log reduction, X is COD and m, n are reaction rate constants) was able to
successfully predict the disinfection kinetics of TiO2 in the presence of organic matter (R2
= 0.944). In the next study, TiO2 coatings having a thickness of 50-100 µm were
developed on stainless steel substrates either by dip-coating or painting. Among several
tested coating formulations using different binders; TiO2 coatings containing shellac,
polyurethane, and polycrylic as binders at 4 to 16 weight percent were physically stable
when subjected to adhesion strength, scratch, and wear resistance tests. An indented
coupon technique was found to be the most appropriate method to determine the
bactericidal property of TiO2 nanocoatings. TiO2 coating with polycrylic showed the
greatest reduction followed by TiO2 coating with polyurethane, and shellac. On repeated
use of coatings for 1, 3, 5, and 10 times, TiO2 coating with polycrylic was found to be
physically more stable and able to retain its original bactericidal property. The results of
this research show promise to development of durable photocatalytic antimicrobial
nanocoatings on food contact surfaces to help ensure a safe food processing environment.
INDEX WORDS: Titanium dioxide, Nanoparticles, Food contact surface, Antimicrobial
coating, Physical stability, Photocatalytic activity, Organic matter, E.coli O157: H7.
EFFICACY OF PHOTOCATALYTIC NANOCOATINGS ON FOOD CONTACT
SURFACES TO INACTIVATE PATHOGENIC MICRORGANISMS
by
VEERACHANDRA K. YEMMIREDDY
B. Tech., Osmania University, India, 2004
M. S., University of Georgia, 2011
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in partial
Fulfillment of the Requirements for the Degree
DOCOTOR OF PHILOSOPHY
ATHENS, GEORGIA
2015
© 2015
Veerachandra K. Yemmireddy
All Rights Reserved
EFFICACY OF PHOTOCATALYTIC NANOCOATINGS ON FOOD CONTACT
SURFACES TO INACTIVATE PATHOGENIC MICRORGANISMS
by
VEERACHANDRA K. YEMMIREDDY
Major Professor: Yen-Con Hung
Committee: Yiping Zhao
Joseph F. Frank
Jennifer L. Cannon
Alexander M. Stelzleni
Electronic Version Approved:
Julie Coffield
Interim Dean of the Graduate School
The University of Georgia
May 2015
iv
DEDICATION
To my parents and my teachers who all have made me what I am today
v
ACKNOWLEDGEMENTS
I would like to thank everyone who has been part of this project. I want to
especially thank my major professor, Dr. Yen-Con Hung, who not only supported me in
successful completion of this research but also encouraged and challenged me throughout
my academic program towards the best realization of my goals. The knowledge and the
training that I obtained under his tutelage is invaluable in shaping me up as a researcher.
Dr. Hung, it was a pleasure to work with you and an honor to have you as my mentor
during my graduate studies at UGA.
I would like to thank Dr. Yiping Zhao for being part of my committee and always
challenging me with questions that helped me to better understand the subject of
photocatalytic nanomaterials. I also would like to thank Dr. Frank, Dr. Cannon, and Dr.
Stelzleni for serving on my Ph.D. committee and guide me through this process. I am
deeply thankful to Mr. Glenn Farrell for all his technical help throughout this research. I
acknowledge the support of all my labmates in Dr. Hung’s research group without whom
this could not have been done. In addition, I would like to extend my thanks to all the
personnel in the Melton building for their friendship and support throughout my research
work at UGA-Griffin campus.
Finally, I want to thank my family and friends, especially my father Mr. Linga
murthy, my mother Mrs. Vijaya lakshmi, my brother Mr. Ramesh kumar, and my wife
Srividya for their love, encouragement, and support.
vi
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS……………………………………………………...…........v
LIST OF TABLES ………………………………………………………………………vii
LIST OF FIGURES …………………………………………………………………….viii
APPENDICES……………………………………………………………………………x
CHAPTER
1 INTRODUCTION…………………………………………………………………….1
2 LITERATURE REVIEW……………………………………………………………..5
3 SELECTION OF PHOTOCATALYTIC BACTERICIDAL TITANIUM DIOXIDE
(TiO2) NANOPARTICLES FOR FOOD SAFETY APPLICATIONS……..…….....85
4 EFFECT OF FOOD PROCESSING ORGANIC MATTER ON
PHOTOCATALYTIC BACTERICIDAL ACTIVITY OF TITANIUM DIOXIDE
(TiO2) ...……………………………………………..……………………………...109
5 METHOD DEVELOPMENT FOR CREATING TITANIUM DIOXIDE (TiO2)
NANOCOATINGS ON FOOD CONTACT SURFACES AND METHOD TO
EVALUATE THEIR DURABILITY AND PHOTOCATALYTIC BACTERICIDAL
PROPERTY ……………………………………………………………………….139
6 EFFECT OF BINDER ON THE PHYSICAL STABILITY AND BACTERICIDAL
PROPERTY OF TITANIUM DIOXIDE (TiO2) NANOCOATINGS ON FOOD
CONTACT SURFACES ………………………………………………………….172
7 CONCLUSIONS ……………………………………………….............................201
vii
LIST OF TABLES
Table 3.1. Characteristics of commercial TiO2 NPs………………………….………...104
Table 3.2. Effect of light intensity and volume on bactericidal activity of TiO2 NPs….105
Table 4.1. Comparison of kinetic models to predict the TiO2 disinfection efficacy with or
without organic matter………………………….………………………………………131
Table 4.2. Effect of pH of wash solution containing organic matter on the bactericidal
activity of TiO2…………………………………………………………………………132
Table 4.3. Comparison of fitted isotherm parameters of empirical model……………..133
Table 5.1. Composition of different TiO2 nanocoatings………………………………..164
Table 5.2. ASTM D3359-02 classification of adhesion test results……………………165
Table 5.3. Physical stability results of TiO2 nanocoatings with different binders……..166
Table 5.4. Bactericidal activity of TiO2 nanocoatings using different test methods.….167
Table 6.1. Details of the binders and the composition of different TiO2 nanocoatings..195
Table 6.2. Estimated surface coverage of nanocoatings with the binder and the TiO2
nanoparticles……………………………………………………………………………196
Table 6.3. Physical stability of TiO2 coatings before and after repeated use
experiment……………………………………………………………………………...197
Table B1. Summary of photocatalytic bactericidal activity of various types of Fe2O3 in
suspension and as a nanocoating……………………………………………………….220
viii
LIST OF FIGURES
Fig 2.1. Semiconductor photocatalysis…………………………………………………..83
Fig 2.2. TEM of a dispersion of TiO2 Degussa P-25 (1 mg/L) in contact with E. coli K-12
Cells…………………………………………………………...…………………………84
Fig 3.1. Schematic of photocatalytic disinfection set-up……………………..………...106
Fig 3.2. Effect of TiO2 source and bacterial cell harvesting conditions on the log
reduction………………………………………………………………………………..107
Fig 3.3. Comparison of photocatalytic degradation of methylene blue and photocatalytic
disinfection rate of E.coli O157:H7 among different TiO2 NPs……………………….108
Fig 4.1. Effect of different levels of organic matter from produce and meat extract
solutions on the log reduction of E.coli O157:H7 by TiO2 photocatalysis…………….134
Fig 4.2. Effect of turbidity of produce and meat extract solutions on the log reduction
of TiO2 of E.coli O157:H7 by TiO2 photocatalysis for 3h ……………………………135
Fig 4.3. Correlation between total phenolics and COD of produce extract as well as total
phenolics and log reduction of E.coli O157:H7 by TiO2 photocatalysis ……………..136
Fig 4.4. Correlation between protein content and COD of meat extract as well as protein
and log reduction of E.coli O157:H7 by TiO2 photocatalysis ………………………..137
Fig 4.5. Relationship between COD of produce and meat organic matter extracts and
the log reduction of E.coli O157:H7 by TiO2 photocatalysis ……………………..…138
Fig 5.1. Images of TiO2 nanocoatings with shellac (A), polyurethane (B), and polycrylic
(C) binders at different NP to binder concentrations…………………………………168
ix
Fig 5.2. Scanning electron micrographs of the surface of TiO2 coatings with binders
A, B, and C at different NP to binder concentrations………………..…………………169
Fig 5.3. SEM image of TiO2 coating with binder C at 1:8 NP to binder weight ratio (TC8)
showing regions of binder, surface exposed TiO2 NPs, and unexposed TiO2 NPs that are
partly covered by the binder……………………………..………………………..……170
Fig 5.4. In-house fabricated wear resistance tester……………………..………….…...171
Fig 6.1. Effect of type and concentration of binder on the log reduction of E.coli
O157:H7 by TiO2 nanocoatings at 0.5 mW/cm2 UV-A light intensity for 3 h…………198
Fig 6.2. Effect of UVA light intensity on the log reduction of E.coli O157:H7 by TiO2
nanocoatings……………………………………………………..……………………..199
Fig 6.3. Bactericidal activity of different TiO2 nanocoatings against E.coli O157:H7
before and after repeated use experiment…………………………...………………….200
Fig A1. TiO2 nanocoating on stainless steel surface (SS) using (a) Direct coating,
(b) Layer-by-Layer coatings methods……………………………..…………………..210
Fig A2. Effect of coating method on bactericidal activity of TiO2 coatings……….….211
Fig A3. Comparison of TiO2 nanocoatings with binders A (TA8/AT8), B (TB8/BT8),
and C (TC8/CT8) at 1:8 NP to binder weight ratio created by (i) Direct coating method
(TA8, TB8, and TC8), and (ii) Layer-by-Layer coating method (AT8, BT8, and
CT8)………………………………………………………………………………….212
x
APPENDICES
A. STRATAGIES TO IMPROVE PHOTOCATALYTIC BACTERICIDAL
PROPERTY OF TiO2 NANOCOATINGS………………………………………204
B. STUDIES ON BACTERICIDAL ACTIVITY OF VISIBLE LIGHT ACTIVATED
IRON OXIDE (Fe2O3) NANOPARTICLES AND NANOCOATINGS….……..213
1
CHAPTER 1
INTRODUCTION
Surface cross-contamination of foodborne pathogens to food products during
processing or preparation is a major concern to both consumers and food manufacturers
alike. Goddard (2011) stated that “As food production becomes increasingly automated,
the number of surfaces with which foods comes into contact and the subsequent potential
for contamination increases”. Several studies in the past have demonstrated that both
food contact and non-food contact surfaces are major source of microbial cross-
contamination. In general, hygienic processing is assured by the implementation of
cleaning and disinfection operations adapted to the process using different physical,
chemical, and mechanical procedures. Sanitation of food processing equipment is a
regular practice, but in some cases, conventional cleaning and disinfection operations
may be insufficient to achieve satisfactory decontamination (Meylheuc et al, 2006). It is
reported that bacteria may develop resistance to some disinfectants, and it has been
suggested that different types of disinfectants should be used alternately to prevent
establishment of resistant house strains (Doyle, 2005). In addition, several chemical
disinfection methods are well known for generating toxic disinfection by-products. In this
context, advanced oxidation processes involving photocatalytic nanomaterials have
shown great promise as effective non-targeted disinfectants for wide range of
microorganisms and chemical contaminants.
2
Photocatalysis is a versatile and effective process that can be adapted for use in
many disinfection applications (Gamage et al, 2010). Over the last decade, there is an
increased interest in the application of photocatalytic semiconductor nanoparticles (NPs)
for the purpose of food safety and quality enhancement. Of the various photocatalytic
NPs tested to date, Titanium dioxide (TiO2) has been recognized as the most promising
photocatalyst. Heterogeneous photocatalysis using TiO2 is a safe, non-hazardous, and
ecofriendly process which does not produce any harmful by-products (Lan et al, 2013).
TiO2 NP embedded coatings have shown great promise as effective disinfectants over a
range of microorganisms. However, majority of the past research did not fully address the
problem of durability of these coatings on usage. Application of this technology on food
contact surfaces is required to address crucial aspects of stability of these coatings and
migration or release of NPs into the food systems. However, with appropriate binding
agents, stable and permanent TiO2 nanocoatings with strong bactericidal property can be
developed. Hence, the overall goal of this project was to create physically stable and
durable TiO2 nanocoatings on food contact surfaces and evaluate their photocatalytic
bactericidal property. Specific objectives include:
1. To identify most efficient TiO2 NPs with strong bactericidal property and
determine the optimum conditions for their photocatalytic activity.
2. To determine the effect of food processing organic matter on the
photocatalytic bactericidal activity of TiO2 NPs identified from Objective 1.
3. To develop a method to create TiO2 nanocoatings on stainless steel surfaces
using different binding agents.
3
4. To determine the physical stability and photocatalytic bactericidal property of
TiO2 nanocoatings developed from Objective 3.
This dissertation is divided into total seven chapters. The first chapter presents an
introduction and rationale on which the dissertation is based, including specific
objectives. The second chapter presents the literature review on topics such as microbial
food safety concerns, nanotechnology based intervention strategies, synthesis and
characterization of nanocoatings, antimicrobial activity of TiO2 nanomaterials, safety and
toxicity issues of NPs. The third chapter investigates the effect of different variables on
the photocatalytic bactericidal property of several commercial TiO2 NPs in suspension
and identifies the most efficient TiO2 NPs to create nanocoatings. The fourth chapter
presents the effect of food processing organic matter on bactericidal property of TiO2
NPs that are identified from the study in chapter three. The fifth chapter is based on a
study which developed a method to create TiO2 nanocoatings using different binding
agents on stainless steel substrates. In addition, this study also presents the methods to
evaluate physical stability and bactericidal property of TiO2 nanocoatings. The sixth
chapter describes the effect of different binding agents on durability and bactericidal
property of TiO2 nanocoatings. Finally, the seventh chapter outlines overall conclusions
of the research carried out in this project.
4
References:
Doyle, E. M. (2005). Food antimicrobials, cleaners, and sanitizers, Food Research
Institute, UW-Madison.
http://fri.wisc.edu/docs/pdf/Antimicrob_Clean_Sanit_05.pdf. (Accessed on
November 2012).
Gamage, J., & Zhang, Z. (2010). Applications of Photocatalytic Disinfection.
International Journal of Photoenergy. Article ID 764870,
doi:10.1155/2010/764870, 1-11.
Goddard, J. M. (2011). Improving the Sanitation of Food Processing Surfaces. Food
Technology, 65(10), 40-45.
Lan, Y., Lu, Y., & Ren, Z. (2013). Mini review on photocatalysis of titanium dioxide
nanoparticles and their solar applications. Nano Energy, 2(5), 1031-1045.
Meylheuc, T., Renault, M., & Bellon-Fontaine, M. N. (2006). Adsorption of a
biosurfactant on surfaces to enhance the disinfection of surfaces contaminated
with Listeria monocytogenes. International Journal of Food Microbiology, 109(1-
2), 71-78.
5
CHAPTER-2
LITERATURE REVIEW
PHOTOCATALYTIC ANTIMICROBIAL NANOCOATINGS IN FOOD SAFETY-
PAST RESEARCH, PRESENT STATUS AND FUTURE PROSPECTS
6
INDEX
I. Microbial Food Safety Concerns Page #
1 Microbial cross-contamination and its impact on food
safety
7
2 Major sources of microbial cross-contamination 7
3 Role of shiga toxin-producing Escherichia coli in cross-
contamination
9
4 Methods of disinfection and sanitation 10
II. Nanotechnology Based Intervention Strategies
1 Nanotechnology & its applications in food safety 11
2 Antimicrobial nanoparticles 13
3 Photocatalytic nanoparticles 22
4 Principle and mechanism of photocatalysis 23
5 Applications of TiO2 photocatalysis 26
6 Mechanism of TiO2 antimicrobial activity 28
III. Synthesis and Characterization of Nanocoatings
1 Methods to synthesize nanostructured materials 31
2 Methods to develop antimicrobial nanocoatings 32
3 Methods to evaluate surface characteristics and physical
stability of nanocoatings
46
IV. Antimicrobial Activity of TiO2 Nanomaterials 1 Bactericidal activity of TiO2 in suspension vs coating 48
2 Studies related to bactericidal activity of TiO2 for food
safety applications
51
3 Considerations for testing antimicrobial activity of
photocatalytic nanomaterials
53
V. Safety concerns on use of NPs
1 Toxicity issues 58
2 Regulatory framework 60
VI. Knowledge Gap 1 Research scope 62
7
I. MICROBIAL FOOD SAFETY CONCERNS
1. Microbial cross-contamination and its impact on food safety
Microbial cross-contamination is a general term which refers to the direct or
indirect transfer of bacteria or virus from a contaminated product to a non-contaminated
product through various routes. A survey conducted by the World Health Organization in
Europe indicated that almost one quarter of the total foodborne outbreaks are closely
associated with microbial cross-contamination events involving contaminated equipment,
unhygienic processing, contamination through food handlers and inadequate storage
conditions (WHO, 1995). The US Centers for Disease Control and Prevention (CDC)
reported that 19% of foodborne diseases caused by bacteria in the years between 1993 to
1997 in the United States were associated with contaminated equipment and poor hygiene
practices, respectively (IFT, 2004). Similarly, the UK outbreak surveillance system
reported that cross-contamination was the main contributing factor (32%) in the
outbreaks investigated in the period 1999-2000 (WHO, 2003). In addition, several
unaccounted cases of microbial cross-contamination go unnoticed severely impacting the
safety of global food supply chain.
2. Major sources of microbial cross-contamination
Several studies in the past have demonstrated that the microorganisms on the
surfaces in food processing plants are an important source of product contamination and
may lead to food spoilage as well as transmission of disease (Meylheuc et al., 2006;
Chasseignaux et al., 2002; and Salvat et al., 1995). In addition, food residues that
accumulate on inert structural surfaces, such as floor drains, conveyors, and product tote
boxes can act as continuous culture systems in which microorganisms reside and multiply
8
to form mature biofilms that are hard to remove by regular sanitation protocols (Bower et
al, 1996). Surface contamination, colonization, and subsequent biofilm formation events
have been implicated in several foodborne diseases and outbreaks (de Valk et al, 2001).
De Boer and Hahne (1990) showed the ease with which Salmonella can be
transferred from chicken to utensils, a variety of kitchen surfaces, hands, and other foods;
from those surfaces Salmonella cells were recovered up to 6 h after contamination.
Another study by Cogan et al (1999) reported that in kitchens where chicken had been
prepared, the prevalence of Salmonella was 60 % for cutting boards and 10 % for door
handles, cupboards, ovens, sink-rims and refrigerators. On the other hand, minimally
processed foods satisfy the growing consumer demand for more natural, fresh, and highly
nutritious foods with a lower amount of preservatives. However, minimally processed
foods may suffer a high risk of cross-contamination from the processing environment and
equipment, cutting boards, knifes or the working surfaces (Carrasco et al, 2012).
Similarly, microbial cross-contamination is a serious risk in ready-to-eat (RTE) foods as
well. Studies conducted on RTE products revealed that factors such as food handlers,
aprons, utensils, and work surfaces are potential sites for bacterial contamination (Pal et
al, 2008; Christison et al, 2007; Lues and Van Tonder, 2007; Lunden et al, 2002).
Several multi-state foodborne outbreaks in the United States such as an ice-cream
premix contaminated with Salmonella enteritidis (Hennessy et al, 1996), peanut butter
contaminated with Salmonella tennessee (Chang et al, 2013), and cantaloupes
contaminated with Listeria monocytogenes (McCollum et al, 2013) were ultimately
traced back to either contaminated equipment surfaces or unhygienic processing and
preparation, packaging, and transportation conditions. This shows that both food contact
9
and non-food contact surfaces pose high risk of microbial cross-contamination seriously
affecting the public health.
3. Role of shiga toxin-producing Escherichia coli in cross-contamination
Shiga toxin-producing Escherichia coli (STEC) strains of various serotypes are
important foodborne pathogens that pose a serious public health concern, resulting in
significant financial burden. It has been estimated that E.coli O157:H7 is responsible for
over 73,000 cases of illness each year in the United States (Wang et al, 2012). It is
reported that various STEC serotypes have the ability to attach, colonize, and form
biofilms on a wide variety of food contact surfaces commonly used in meat processing
plants as well as on vegetables and meat products (Silagyi et al, 2009). A wide variety of
materials commonly used for food processing equipment such as stainless steel,
aluminum, nylon, Teflon, rubber, plastic, glass, and polyurethane were found to become
ideal hosts for STEC biofilms (Silagyi et al, 2009). It is also reported that STEC may
form biofilms in different areas of food processing environments, such as floors, walls,
pipes, and drains, etc. (Marouani-Gadri et al, 2010). In particular, O157:H7 strains were
found to form biofilms most efficiently on stainless steel and on high density
polyethylene surfaces; and O157:H7 biofilms on stainless steel were able to transfer the
bacteria to meat, poultry, and other food products (Silagyi et al, 2009; Stopforth et al,
2003).
Dourou et al (2011) reported that the contamination of beef carcasses with E. coli
0157:H7 may occur during the slaughtering, dressing, chilling or cutting stages of
processing. As a consequence, there is a potential for E.coli O157:H7 population to be
distributed to the surface of equipment associated with slaughter and fabrication and the
10
environment via aerosols or direct contact, and potentially contaminate unadulterated
carcasses and fresh meat products. For several of the outbreaks the cause is believed to be
lack of or insufficient cleaning and disinfection of equipment and surfaces contaminated
with pathogens (Moretro et al, 2012). For example, contaminated onions due to poor
cleaning and sanitizing of equipment were likely cause of a pathogenic E. coli 0157:H7
outbreak at a fast–food restaurant in Canada that sickened 235 people (NCCE, 2009).
Strong attachment of the STEC biofilms on the food surfaces may also affect the
efficiency of antimicrobial interventions applied to food products for reducing
contamination. Thus, the contamination of STEC strains and subsequent biofilm
formation pose serious threat in process hygiene and may become a source of cross-
contamination in the food processing environment.
4. Methods of disinfection and sanitation
Normally, hygienic processing is assured by implementation of cleaning and
disinfection operations adapted to the process using different physical, chemical, and
mechanical procedures. Most commonly, several alkaline detergents, chlorinated
compounds, iodophors, encapsulated lysozyme, peroxyacetic acid, quarternary
ammonium compounds, electrolyzed water, and numerous other commercial disinfectants
are used for this purpose (Doyle, 2005). Alternative methods, such as ozonation,
irradiation, high pressure water washing, and fumigation were also found to be effective.
Sanitation of food processing equipment is a regular practice, but in some cases,
conventional cleaning and disinfection operations may be insufficient to achieve
satisfactory decontamination (Meylheuc et al, 2006). In addition, several chemical
disinfection methods are well known for generating toxic disinfection byproducts. Other
11
treatment methods such as irradiation have their own problems and limitations, such as
lack of residual effect and generating small colony variants (Doyle, 2005). This indicates
that merely relying upon certain established sanitation and disinfection techniques may
not be sufficient to address emerging problems. Modification of surfaces with
antimicrobial agents to prevent the growth of harmful microorganisms has received much
attention for application in biomedical devices and health as well as in the food and
personal hygiene industries (Rai et al, 2010). Such antimicrobial coatings are required to
have long lasting efficacy, ease of fabrication, and no toxicity for effective use in food
safety applications. In this context, nanotechnology based advanced oxidation processes
involving photocatalytic nanoparticles (NPs) have shown great promise as an effective
non-targeted disinfectants for a wide range of microorganisms and chemical
contaminants.
II. NANOTECHNOLOGY BASED INTERVENTION STRATEGIES
1. Nanotechnology & its applications in food safety
The concept of nanotechnology was first introduced by Richard Feymann in 1959
at a meeting in American Physical Society (Khademhosseini and Langer, 2006). Since
then, nanotechnology has developed into a multidisciplinary field of applied science and
technology. Nanotechnology is the ability to work on a scale of about 1 to 100 nm (1m =
109 nm) in order to understand, create, characterize, and use material structures, devices,
and systems with new properties derived from their nanostructures (Roco, 2003).
Nanomaterials are characterized by having at least one dimension in this size range (i.e. 1
to 100 nm), although the upper limit of 100 nm is used by general consensus in many
cases the nano-properties still exist beyond this size limit (Rossi et al, 2014). At this
12
nanoscale, the surface-to-volume ratios of materials become large and their electronic
energy states become discrete, leading to unique physic-chemical, electronic, optical,
magnetic, mechanical, and biological properties which can be manipulated suitably for
desired applications (Rai and Bai, 2012).
The phenomenon that takes place at the nanometer scale offers lots of
opportunities for innovation that have the potential to impact global food supply.
Nanotechnologies can be applied in the entire food chain, from production to processing,
product safety, packaging, transportation, storage, and delivery (Cushen et al, 2012;
Silvestre et al, 2011; Weiss et al, 2006). While nanotechnology has revolutionized the
fields of medicine, electronics, energy, and defense, its application in food sector is
relatively new, as most of the research in this area is in its infancy. In the last one decade,
several excellent reviews have been written discussing the potential benefits of
nanotechnology in food sector (Rossi et al., 2014; Sastry et al., 2013; Cushen et al., 2012;
Morris., 2011; Chen and Yada, 2011; Duncan and Timothy, 2011; Brody., 2006; Chen et
al., 2006; Moraru et al., 2003). One promising area of such application is the use of
nanotechnology based intervention strategies for food safety and quality.
Many applications, including food production and storage might benefit from the
incorporation of safe, economical, and wide spectrum long-lasting biocides into
polymers, paints, or working surfaces (Fernandez et al, 2008). For the last one decade,
there is an increased interest in the application of photocatalytic disinfection techniques
for the purpose of food safety and quality enhancement. Certain metal ions, such as
copper, silver, zinc, palladium, titanium, or iron, occur naturally and can be used for
novel food safety measures. These materials are recognized to have no adverse effects on
13
eukaryotic cells below certain concentrations (Ibhadon et al, 2013), thus being excellent
candidates for the implementation of novel strategies in food safety by incorporating in
food contact substances. As per the US FD&C Act (Section 409, US FDA,1998a) a food-
contact substance can be defined as "any substance that is intended for use as a
component of materials used in manufacturing, packing, packaging, transporting, or
holding food if such use is not intended to have a technical effect in such food". Common
types of food contact substances include coatings, plastics, paper, adhesives, as well as
colorants, antimicrobials, and antioxidants found in packaging. Hence, NPs can also be
used to develop antimicrobial coatings on food contact and non-food contact surfaces to
provide additional layer of protection along with the existing sanitizers and disinfectants
to ensure safe food processing environment.
2. Antimicrobial nanoparticles
The antimicrobial agents currently used in the food industry can be classified into
two categories: i) organic, and ii) inorganic. The key advantages of inorganic
antimicrobial agents, when compared to their organic counterparts, are improved safety
and stability at high temperature and pressures (Fu et al., 2005; Sawai, 2003). Similarly,
nanomaterials can be grouped into two main categories: i) Organic which include carbon
NPs such as fullerenes, and (ii) inorganic NPs such as those of nobel metals (gold, silver,
and platinum), magnetic NPs (iron, cobalt, and nickel), and those of semiconductor NPs
(oxides of titanium, zinc, and cadmium etc.). Inorganic NPs have gained significant
importance due to their ability to withstand adverse processing conditions without losing
their original characteristics. Currently, a variety of NPs have been explored for their
14
potential antimicrobial properties. These include NPs of silver, gold, zinc, silica,
aluminum, copper, magnesium, titanium, iron and their respective oxide forms.
The antimicrobial activity of the NPs is known to be a function of the surface area
in contact with the microorganisms. The small size and large surface area (i.e. high
surface to volume ratio) of the NPs enhances their interaction with the microbes to carry
out a broad range of probable antimicrobial activities (Rai and Bai, 2012). Inorganic NPs
with antimicrobial activity when embedded and coated onto the surface can find immense
applications in water treatment, food processing, and packaging. Therefore, the use of
inorganic antimicrobial agents as a coating on food processing equipment and other food
contact and non-food contact surfaces to reduce the chances of microbial cross-
contamination has attracted much attention. The reported antimicrobial properties of
some of these NPs were briefly described here:
2.1. Silver
Among inorganic antibacterial agents, silver (Ag) has been used extensively for
very long time to control spoilage and fight infection (Chou et al., 2005). However, the
mechanism of inhibitory action of silver ions and silver NPs on microorganisms is not
well established until now. Several possible mechanisms have been proposed for
antimicrobial property of silver.
It is assumed that the high affinity of silver towards sulfur and phosphorus is the key
element of the antimicrobial effect. Due to the abundance of sulfur-containing proteins on
the bacterial cell membrane, silver NPs can react with sulfur-containing amino acids
inside or outside the cell membrane, which in turn affects bacterial cell viability. It was
also proposed that the Ag+ ions released from Ag NPs can interact with phosphorus
15
moieties in DNA, resulting in the obstruction of DNA replication and inhibition of
enzyme functions in the bacterial cell (Matsumura et al, 2003; Gupta and Silver, 1998).
Another proposed phenomena is that the Ag NPs adhere to the cell surface degrade the
lipopolysaccharides and eventually form pits in the membranes, largely increasing the
cell permeability and eventual death (Sondi and Salopek-sondi, 2004). Rai et al (2009)
provided more detailed review on antimicrobial mechanism of silver NPs.
The antibacterial and antiviral activity of silver, silver ion, and silver compounds
have been thoroughly investigated. Physico-chemical properties such as size, shape, and
concentration of NPs play an important role in the antimicrobial activity of silver. Gogoi
et al (2006) reported that the Ag NPs with size less than 10 nm are more toxic to bacteria
such as E.coli and P. aeruginosa. Pal et al (2007) reported that triangular silver
nanoplates containing more reactive planes were found to be more toxic than silver
nanorods, spheres, or Ag+
ions. Thus, the silver NPs exhibit a shape-dependent
interaction with the bacterial cells. Araujo et al (2012) found that increasing the
concentration of Ag NPs from 6 to 60 µg/mL in the suspension increased the
antimicrobial activity. Kim et al (2008) found that silver ions were also photoactive in the
presence of UV-A and UV-C irradiation, leading to enhanced UV inactivation of bacteria
and viruses.
The most common nanocomposites used as antimicrobial films for food
packaging are based on silver, which is well known for its strong toxicity to a wide range
of microorganisms (Liau et al, 1997) with high temperature stability and low volatility
(Kumar and Munstedt, 2005). Though, silver has been reported as an excellent
antimicrobial agent, its mode of action, dose required for killing the microorganisms in
16
food systems and reported toxic effects limits its usage in food applications (Fernandez et
al 2010). It has been demonstrated that silver is non-toxic to humans cells at lower
concentration. However, high concentrations of silver are required to exert antimicrobial
activity in food systems limiting the feasibility of using silver widely in food safety
applications. For example, Llorens et al (2012) reported that a concentration of 60 mg
Ag+/kg was necessary to reduce the microbial load of 1-log CFU/mL in absorbent pads in
contact with beef meat. They also reported that the natural chelating agents, especially
proteins, counteract the antimicrobial activity of silver ions.
2.2. Gold
Gold (Au) NPs are known to be the most stable NPs and can be engineered to
possess excellent chemical or photo-thermal properties (Rai and Bai, 2012). A detailed
review on chemistry, properties, catalytic and biological applications of Au NPs was
provided by Daniel and Astruc (2004). Photocatalytic gold NPs conjugated with specific
antibodies and antibiotics was found to exhibit excellent antibacterial activity over a
range of Gram-positive and Gram-negative bacteria. Rai et al (2010) reported that
antibiotic cefaclor reduced gold NPs have potential bactericidal activity against S. aureus
and E. coli. They reported that the antibiotic inhibits the synthesis of peptidoglycan layer,
making cell walls more porous and the gold NPs generate holes in the cell wall, resulting
in the leakage of cell contents and eventual cell death. It may also be possible that gold
NPs bind to the DNA of bacteria and inhibit the uncoiling and transcription of DNA thus
promoting the death of bacteria. In another study by Perni et al (2009), polymers
containing methylene blue and gold NPs showed a reduction of up to 3.5 log for
methicillin-resistant S. aureus and E. coli in 5 min when exposed to low power laser light
17
(660 nm). It is believed that the bactericidal activity is due to the light-induced
production of singlet oxygen and other reactive oxygen species (ROS) generated by
methylene blue in presence of gold NPs. They further reported that the presence of gold
NPs enhanced the hydrophobic properties of the polymer as well as its bactericidal
activity. This shows that gold NPs can be used to develop antimicrobial coatings on
different surfaces. However, the feasibility of Au NPs for use in practical applications is
very limited due to cost.
2.3. Copper oxide
Copper oxide (CuO) is another semiconductor metal oxide with a photocatalytic
property. Compared to other NPs, copper oxide is cheap, stable and mixes well with
polymers making it an attractive nanomaterial for a wide range of applications (Rai and
Bai, 2012). CuO was found to be effective in killing a range of bacterial pathogens
involved in hospital-acquired infections. However, like silver NPs, a high concentration
of CuO is required to achieve significant bactericidal effect (Ren et al, 2009). Several
mechanisms have been proposed for antimicrobial activity of Cu/CuO. However, the
exact mechanism behind bactericidal effect of copper NPs is still unclear. One possible
mechanism is that the metallic and ionic forms of copper produce hydroxyl radicals that
damage essential proteins and disrupt the mechanism of DNA replication in bacterial
cells. Studies reported that due to greater abundance of amines and carboxyl groups on
the cell surface of B. subtilis, it has high affinity towards CuO NPs and more susceptible
to inactivation by CuO. Also, presence of copper ions inside bacterial cells disrupts their
biochemical processes (Rai and Bai, 2012). The antimicrobial activity of copper NPs
18
depends on the combination of several factors such as shape, concentration, pH,
temperature, and the concentration of bacteria.
In a study conducted on antibacterial activity of different NPs, CuO was found to be the
most toxic against E. coli, B. subtilis, and S. aureus followed by ZnO, NiO, and Sb2O3
(Baek and An, 2011).
Studies have been conducted to assess the potential of CuO NPs embedded in a
range of polymer materials. A lower contact-killing ability was observed in comparison
with release killing ability against MRSA strains. This suggests that a release of Cu ions
into the local environment is required for optimal antimicrobial activity (Ren et al, 2009;
Cioffi et al, 2005). Noyce et al (2006) reported a diminished risk associated with E. coli
O157:H7, when food processing work surfaces were coated with copper cast alloys.
However, the presence of beef residues found to be a limiting factor for the required
growth inhibition.
2.4. Zinc oxide
Zinc oxide (ZnO) NPs have been used in sunscreens, coatings, and paints due to
their high UV absorption efficiency and transparency to visible light (Franklin et al,
2007). ZnO NPs exhibit strong antibacterial activities on a broad spectrum of bacteria
(Sawai, 2003; Adams et al, 2006; Jones et al, 2008). Even though the antibacterial
mechanism of ZnO is not well understood, the photocatalytic generation of hydrogen
peroxide was proposed to be one of the primary mechanisms (Sawai, 2003). In addition,
penetration of the cell envelope and disorganization of bacterial membranes upon contact
with ZnO NPs were also found to inhibit bacterial growth (Huang et al, 2009).
Contrasting results have been reported regarding the effect of particle size on the
19
antibacterial activity of ZnO. Jones et al (2008) observed that smaller ZnO particles were
more toxic than bigger particles, but no size related effect was found in another study by
Franklin et al (2007). Li et al (2011) studied the potential use of nano-packaging
containing ZnO NPs during the storage of Fuji apple cuts. They observed a better
retention of quality indicators such as ascorbic acid and polyphenol content, and lower
counts of typical altering microorganisms. However, reports suggest that ZnO suffers
from photo-corrosion problems upon excitation in the solution.
2.5. Magnesium oxide
Stoimenocv et al (2002) reported that reactive magnesium oxide (MgO) NPs and
halogen (Cl2, Br2) adducts of these NPs both in the form of dry powder and in water
slurries are very effective against Gram-negative (E.coli) and Gram-positive (B.
megaterium) bacteria as well as spores (B. subtilis). However, spores were found to be
less susceptible to the action of MgO compared to vegetative cells. MgO NPs with the
crystal size less than 10 nm exhibit high bactericidal activities since their high surface
area, presence of defective sites and positive charges on surface exhibit strong affinity
towards electronegative bacteria and spores. Further, the extremely small size of MgO
allows many particles to cover bacterial cells to a high extent and bring high
concentration of halogen in an active form in proximity to the cell. Similarly, Lin et al
(2005) demonstrated that γ -Al2O3 with highly dispersed MgO on the surface is efficient
bactericide, and the one with the 20% load amount of MgO can kill more than 99%
bacteria and spore cells. Similarly, Huang et al (2005) reported a high bactericidal
efficacy of MgO NPs both when used directly and as an additive in an interior wall paint.
20
2.6. Iron oxide
Magnetic NPs have aroused increased interest for their potential applications in
various fields, such as advanced materials, biomedicine, diagnostics, energy, and the food
sector (Cao et al, 2012). Iron oxide (Fe2O3) exists in different polymorphs such as alpha,
beta, gamma and epsilon. Chirita et al (2009) discussed the physical, photochemical and
photo- electrochemical properties and applications of iron oxide. Iron oxide is
particularly interesting because of its stability against photo/chemical corrosion at neutral
or basic pH and has band gap energy of about 2.0 to 2.2 eV corresponding to the
absorption of 564 to 620 nm light (Basnet et al, 2013). Especially, α- Fe2O3 or hematite
form of iron oxide is known for its useful photocatalytic properties for solar energy
conversion and water splitting. In addition, hematite has shown promise as a disinfectant
under visible light (λ< 552 nm) photocatalysis. Fe2O3 biocidal applications are not widely
reported. Sultana et al (2012) reported 90% reduction of E.coli and S. aureus with a bio-
ceramic material with 7% loading of iron and titanium metal oxide incorporation. Prucek
et al (2011) identified minimum inhibitory concentration for ten different bacterial and
four different fungal strains using composite silver, α- Fe2O3 and Fe3O4 (maghamite)
NPs. Zhang et al (2011) studied adsorption kinetics of α- Fe2O3 at different sizes for
inhibition of E.coli and reported faster inhibition rates at smaller particle sizes. Tran et al
(2010) reported inhibition of S. aurues growth using PVA coated iron oxide NPs. Basnet
et al (2013) reported that sputter deposited Fe2O3 thin films and nanorod coatings on
glass exhibited around 1.5 and 4.5 log reductions of E.coli O157:H7, respectively.
However, photocatalytic disinfection studies conducted by our group with commercial
and chemical/physically synthesized Fe2O3 NPs in suspension has shown less than 0.5 log
21
CFU/mL of E.coli O157:H7 even after prolonged treatment time of about 3 to 4 h.
Though, α-Fe2O3 is low cost, abundant, and has narrow bandgap for harnessing solar
energy, it suffers from rapid charge recombination and a short charge carrier diffusion
length. As a result of these draw backs, renewed interest in this material has focused on
its modification with cationic dopants such as Cr and Mo to improve its charge transport
properties or doping with Si to reduce the charge diffusion path length (Tran et al, 2005).
2.7. Tungsten oxide
Tungsten oxide (WO3) has received renewed interest in the photocatalytic
applications due to its narrow bandgap (2.7 eV). However, like Fe2O3 NPs, Tungsten
oxide, has the disadvantage of a low electron conduction band and rapid charge pair
recombination. Studies reported that coupling WO3 with platinum co-catalyst help to
achieve much lower reduction potentials. This has increased the use of WO3 as one of the
very few highly visible-light-active single-phase oxide photocatalysts (Ibhadon et al,
2013).
2.8. Titanium dioxide
Of the various semiconductor nanomaterials tested to date, titanium dioxide
(TiO2) has been recognized as the most promising photocatalyst because of its unique
electronic band structure, photostability, chemical inertness, low cost, non-toxicity,
commercial availability, and capability of repeated use without substantial loss of
catalytic activity (Lan et al, 2013). Titanium dioxide is the oxide of titanium with a
chemical formula of TiO2 and was first discovered in 1791 from ilmenite. It is also
known as titanium (IV) oxide, titania, titanium white or pigment white 6 in building
paints, and E171 in food coloring (Fujishima and Zhang, 2006). TiO2 mainly exists in
22
three polymorphs namely, anatase, rutile, and brookite. Among these, only anatase and
rutile form of TiO2 were found to show high photocatalytic activity. Anatase possesses an
energy band gap of 3.2 eV with an absorption edge at 386 nm which lies in the near UV
range. Anatase is the most stable form of TiO2 and can be converted to rutile by heating
to temperatures above 700 °C. Rutile has a narrow band gap of 3.02 eV, with excitation
wavelengths that extend into the visible light range (410 nm). However, anatase is
considered as the most photochemically active phase of titania. The reason for this higher
activity can be attributed to the combined effect of the higher surface adsorptive capacity
of anatase and its higher rate of hole trapping. Afterwards, several studies have shown
that mixtures of anatase-rutile or brookite-anatase were more active than anatase alone
(Visai et al, 2011). Degussa P-25 is one commercially available form of TiO2 which
consists of both rutile and anatase phase at around 1:3 weight ratios. Several studies
showed that Degussa P-25 has excellent photocatalytic properties and is used as a
standard to compare the photocatalytic activity of other nanomaterials (Mills et al, 1997).
A more detailed review on synthesis, properties, principles, and applications of TiO2
photocatalysts were reported by Chen et al (2007) and Linsebigler et al (1995). The
bactericidal mechanism and the major applications of TiO2 were further discussed in the
next sections.
3. Photocatalytic nanoparticles
Among inorganic antimicrobial NPs, photo-activated antimicrobial nanostructures
are especially interesting. These photocatalysts include various oxide semiconducting
materials, their metal hybrid nanocomposites, and doped structures such as TiO2, ZnO,
CuO, MgO, CdS, ZnS, SnO2, WO3, SiO2, ZrO2, Fe2O3, Nb2O3, Ag/TiO2, TiO2/CuO,
23
TiO2/Pt, Au/TiO2, Fe2O3/TiO2, and N-, C-, S- doped TiO2 (Fu et al., 2005; Sawai, 2003).
Especially, semiconductor nanomaterials like TiO2, Fe2O3, WO3, and ZnO have enough
band-gap energies to carry-out more efficient photocatalytic reactions. Among these
photocatalytic NPs, the unique electronic band structure of TiO2 makes it as a stand-alone
photocatalyst, and an ideal choice for the photocatalysis. TiO2 is the most frequently used
photocatalyst owing to its photostability and low cost, combined with its biological and
chemical inertness and resistant to chemical corrosion. On the other hand, binary metal
sulfide semiconductors such as CdS and PbS are regarded as insufficiently stable for
catalysis and are toxic. ZnO is also unstable in illuminated aqueous solutions while WO3
and Fe2O3 have been investigated as a potential photocatalysts, but these are generally
less active catalytically compared with TiO2.
4. Principle and mechanism of TiO2 photocatalysis
Semiconductor NPs such as TiO2, ZnO, Fe2O3, CdS, and ZnS can act as
sensitizers for light-induced redox reactions due to their electronic structure, which is
characterized by a filled valence band (VB) and an empty conduction band (CB). Each
semiconductor used as a photocatalyst corresponds to a range of light wavelengths with
which electron hole-pairs may be induced. The size of the band gap of the electron hole-
pairs varies between the semiconductors and band gap is the amount of energy the
semiconductor requires to absorb in order to produce an electron-hole pair. As shown in
the Fig.1, when a photon with energy of hʋ matches or exceeds the band gap energy (Eg)
of the semiconductor, an electron (ecb-), is promoted from the VB, into the CB, leaving a
hole (hvb+) behind. Excited state CB electrons and VB holes can recombine in
picoseconds and dissipate the input energy as heat, get trapped in metastable surface
24
states or react with electron donars and electron acceptors adsorbed on the semiconductor
surface such as molecular oxygen and water. This mechanism generates several types of
reactive oxygen species (ROS) which can be effectively utilized to completely mineralize
organic compounds into CO2 and H2O (Hoffmann et al., 1995).
The band gap energy of anatase form of TiO2 is approximately 3.2 eV, which
effectively means that photocatalysis can be activated by photons with a wavelength of
below 385 nm which falls in UVA region. The absorption of photons with sufficient
energy by TiO2 NPs results in generation of electron (e-) - hole (h
+) pairs as described
before. These conduction band electrons and valence band holes on the surface of TiO2
can react with surface bound O2 or H2O molecules to initiate redox reactions. This
process generates ROS such as hydroxide radicals (OH-), superoxide radicals (O2
-), and
hydrogen peroxide (H2O2) in various chain reactions. The major steps involved in the
photocatalytic oxidation of organic compounds by TiO2 were shown below:
Steps:
1. Photo excitation: TiO2 + hʋ ecb- + hvb
+
2. Electron trapping : ecb- e tr
-
3. Hole trapping : h vb +
h tr +
4. Electron-hole recombination : e-
tr + h+ vb (h
+ tr) e
- cb + heat (non-productive)
5. Oxidation of hydroxyls: H2O + h+ vb
•OH + H
+aq
6. Reduction of oxygen: O2(ads) + ecb - O2
.-
7. Formation of hydrogen peroxide: •OH +
•OH H2O2
8. Chain reaction: O2.- + H2O2
•OH + OH
- + O2
9. Hydroperoxyl radical formation: O2.- + H
+ •OOH
25
10. Mineralization of organic compounds: •OH + Organics+ O2 CO2 + H2O
Furube et al (2001) reported that the trapped charge carriers in steps 1, 2, and 3
are usually TiO2 surface bound and do not recombine immediately after photon
excitation. In the absence of electron scavengers, the photoexcited electron recombines
with the valence band hole in nanoseconds with simultaneous dissipation of heat energy
as shown in step 4. Thus, the presence of electron scavengers is vital for prolonging the
recombination rate and efficient photocatalysis. Presence of an electron scavenger such as
molecular oxygen prevents the recombination of electron-hole pair, and allows the
formation of superoxide radical by following reaction step 6. This superoxide radical can
be further protonated to form the hydroperoxyl radical (Step 9). The hydroperoxyl radical
formed was also reported to have scavenging property and thus, the co-existence of these
radical species can doubly prolong the recombination time of holes in the entire
photocatalytic reaction. However, it should be noted that all these occurrences in
photocatalysis were attributed to the presence of both dissolved oxygen and water
molecules. Without the presence of water molecules, the highly reactive hydroxyl
radicals (Step 5) could not be formed and impede the photodegradation of organic
substances. The h+ tr are powerful oxidants (+1.0 to +3.5 V against NHE), while e tr
- are
good reductant (+0.5 to -1.5 V against NHE), depending on the type of catalysts and
oxidation conditions. The hydroxyl radical is a powerful oxidizing agent, and attacks
organic pollutants present at or near the surface of TiO2. It results in complete
decomposition of toxic and bio-resistant compounds into harmless species such as CO2
and H2O (Step 10).
26
5. Applications of TiO2 photocatalysis
After the initial discovery of photocatalytic water-splitting of TiO2 by Fujishima
and Honda (1972), TiO2 has been widely studied for use in several other applications.
Lan et al (2013) has briefly summarized the photocatalytic applications of TiO2 as
follows:
i. Photocatalytic water-splitting
Fujishima and Honda (1972) first reported the ability of TiO2 to split water into
H2 and O2 in the presence of light having a wavelength shorter than 410 nm. Later,
TiO2 photocatalysis has attracted much attention as one promising method to produce
hydrogen for energy requirements.
ii. Environmental decontamination
Several reviews have been published on environmental decontamination of TiO2
photocatalysts (Ibhadon et al, 2013; McCullagh, 2007). TiO2 photocatalysis can
degrade and mineralize a large variety of environmental contaminants, including
organic and inorganic materials into CO2, H2O, and harmless inorganic anions (Lan et
al, 2013). Photocatalytic properties of TiO2 were effectively used in order to
decompose organic pollutants and purify soil, air, and water (Yu and Brouwers, 2009;
Chaleshtori et al, 2008; McCullagh, 2007).
iii. Photocatalytic disinfection
Since Matasunga et al (1985) first reported the photocatalytic disinfection efficacy
of TiO2 to inactivate bacteria, several studies have been reported the use of TiO2
photocatalysis to kill a wide range of microorganisms. More recently, Foster et al
(2011) reported a comprehensive review on photocatalytic disinfection using TiO2.
27
iv. Photocatalytic self-cleaning surfaces
A TiO2 coated surface becomes superhydrophilic upon irradiation with UV light due
to formation of metastable hydroxyl groups which results in the formation of very thin
closed liquid layer that is characterized by a small contact angle (Watanabe et al, 1999).
This phenomenon supports the cleaning process and enables fast evaporation of the water
film. However, this functionality of TiO2 is reversible and depends on the light exposure
(Wolfrum et al 2002). This photo-induced self-cleaning property of TiO2 has been
extensively used in several applications such as exterior and interior construction
materials, road-construction materials, and household goods etc. (Fujishima and Zhang,
2006). In addition, these properties of TiO2 help to reduce the usage of cleaning agents
and to shorten the cleaning cycles. Especially in the food industry where frequent
cleaning cycles are the norm, this phenomenon could help reduce costs in the long run
(Muranyi et al, 2013).
Heterogeneous photocatalysis using TiO2 is a safe, nonhazardous, and ecofriendly
process which does not produce any harmful by-products. Based on the unique properties
of the photo-induced electron-hole pairs, inertness to chemical environment and long-
term photostability has made TiO2 an attractive material for many commercial
applications, ranging from drugs to foods, cosmetics to catalysts, paints to
pharmaceuticals, and sunscreens to solar cells in which TiO2 is used as a desiccant,
brightener, or reactive mediator (Kamat, 2012). In addition, due to the antibacterial
applications of TiO2-mediated photooxidation, this process shows promise for the
elimination of microorganisms in areas where the use of chemical cleaning agents or
biocides is ineffective or is restricted by regulations, for example in the pharmaceutical
28
and food industries (Skorb et al 2008). Antimicrobial surface coatings based on the
semiconductor TiO2 could provide a positive contribution to maintain the process
hygiene.
6. Mechanism of TiO2 antimicrobial activity
First known bactericidal activity of TiO2 photocatalytic reactions was reported by
Matasunga et al (1985). Since then several important photo-killing mechanisms have
been proposed for TiO2. It is believed that the bactericidal effect of TiO2 is initiated by
the photochemical oxidation of intracellular coenzyme A, which alters the respiratory
activities and leads to eventual death of bacterial cell (Matsunaga et al., 1985, 1988).
Later, Saito et al (1992) reported that a rapid leakage of K+ ions and slow leakage of
RNA and proteins from treated bacterial cells due to TiO2 photocatalytic reaction is the
possible cause for the bactericidal property. A similar mechanism has been suggested by
Hu et al (2007). Another study by Zheng et al (2000) investigated the mechanism of cell
death with a focus on the gross features of cell wall and cytoplasmic membrane damages
caused by TiO2 photocatalytic reactions. Their study measured the hydrolytic rate of
permeability of marker probe (ONPG, a chromogenic substrate) upon reaction with
intracellular β- glycosidase enzyme. This reaction is only possible if there is damage to
the outer membrane and inner membrane through permeation of ONPG towards inside of
the cell or leakage of β- glycosidase towards outside of the cell. Their results suggested
that the initial oxidative damage takes place on the cell wall, where TiO2 photocatalytic
surface makes first contact with intact cell. Cells with damaged cell wall are still viable.
After degradation of outer membrane, oxidative damage proceeds to the underlying
cytoplasmic membrane. This condition results in free efflux of intracellular contents that
29
eventually leads to cell death. Several spectroscopic studies supported this mechanism
and confirm the order of destruction being outer membrane followed by inner membrane
and peptidoglycan layer. Sunada et al (1998), reported photocatalytic degradation of E.
coli endotoxin which is an integral part of the outer membrane. However, there is also
more direct evidence that the lethal action is due to outer membrane and cell wall damage
(Fig. 2). This is mainly due to the production of ROS like hydroxyl radicals (•OH) and
hydrogen peroxide (H2O2) by the photocatalysts under illumination, which can lead to
phospholipid peroxidation and ultimately cell death (Sunada et al., 2003; Cho et al.,
2005). Pigeot-Remy (2012) and Maness et al (1999) demonstrated that lipid peroxidation
can be initiated on cell membrane polyunsaturated phospholipids through TiO2
photocatalytic reaction. Hydroxyl radicals generated by the TiO2 photocatalyst are very
potent oxidants and are nonselective in reactivity. Their findings suggested that OH·, O2-,
and H2O2 generated on the irradiated TiO2 surface resulted in breakdown of cell
membrane structure of E. coli. In contrast, Goginat et al (2006) reported that aggregation
of bacteria onto TiO2 particles is a driving force for the bactericidal effect in suspension.
They observed cytoplasmic membrane started to disintegrate even before illumination.
This NP adsorption alone alters membrane integrity and greatly amplified under
illumination.
It is clear that the first contact between microorganism and ROS occurs on the cell
surface. Therefore, it is the primary target of initial oxidative attack (Maness et al., 1999).
It is assumed that ROS act at different distances. For instance, H2O2 can diffuse into the
solution in contrast to OH radicals which are bound on the surface or react close to it due
to high and unselective oxidation (short lifetime) (Schwegmann et al, 2012). There is a
30
greater consensus that the OH radicals are the primary ROS responsible for the
inactivation of cells (Cho et al, 2005) and hence the physical contact between
photocatalyst and cell have a high impact on the disinfection rate (Goginat al, 2006;
Marugan et al, 2010). The surface interaction of microorganisms with the photocatalyst
during the photo-disinfection is essential for enhancing the inactivation rate. Hence the
transfer of bacterial cell to the close vicinity of the surface generated ROS site remains as
the rate-limiting step in the photocatalytic disinfection.
Induction of oxidative stress due to formation of ROS triggers the NP toxicity and
it depends on various factors such as composition, surface modification, intrinsic
properties of NPs and the bacterial species (Hajipour et al, 2012). In particular, many
previous studies have explored the photogeneration of ROS on the surfaces of metal-
oxide NPs. Hydroxyl radical (·OH) is a strong and nonselective oxidant that can damage
virtually all types of organic biomolecules, including carbohydrates, nucleic acids, lipids,
proteins, DNA, and amino acids. Singlet oxygen (1O2) is the main mediator of the
phototoxicity and can irreversibly damage the treated tissues causing membrane
oxidation and degradation. Although, superoxide anion is not a strong oxidant, it acts as a
precursor for .OH and
1O2. Consequently, these three types of ROS (
.OH, O2
.- and
1O2 )
contribute to the major oxidative stress in biological systems (Li et al, 2012).
Li et al (2012) compared the ROS generation potential of seven types of metal
oxide NPs (nTiO2, nCeO2, nZnO, nCuO, nSiO2, nAl2O3 and nFe2O3) and their bulk
counterparts based on their band energy structures and subsequently analyzed their
antibacterial activity. Bulk particles other than bTiO2 and bZnO did not produce
measurable ROS, whereas all the NPs other than nCuO generated ROS. The average
31
concentration of total ROS (i.e. .OH, O2
.- and
1O2) are in the order of nTiO2 > nZnO >
nAl2O3 > nSiO2 > nFe2O3 > nCeO2 > nCuO and bZnO > bTiO2. Among NPs, nZnO
generated the most O2.-, followed by nFe2O3, nTiO2, and nCeO2, whereas for bulk
materials, only bZnO favors O2.- generation. nTiO2 generated the most
.OH, which was
approximately 2-fold and 6-fold more than that generated by nZnO and nFe2O3. nTiO2
generated the most 1O2, followed by nAl2O3, nZnO, and nSiO2. The enhanced ROS
generation potential of NPs compared to their bulk counterpart is likely due to their large
surface areas, which provide more available reaction sites for light absorption. Other
potentially size dependent properties such as light absorption or scattering, defective
sites, and structural disorder may also lead to the difference in photoactivity.
III. SYNTHESIS AND CHARACTERIZATION OF NANOCOATINGS
1. Methods to synthesize nanostructured materials
Two building strategies are currently used in nanotechnology: a ‘top-down”
approach and the “bottom-up” approach. The commercial scale production of
nanomaterials basically involves the “top-down” approach, in which nanometric
structures are obtained by size reduction of bulk materials, by using milling,
nanolithography, or precision engineering (de Azeredo et al, 2009). The newer “bottom-
up” approach, on the other hand allows nanostructures to be built from individual atoms
or molecules capable of self-assembling (Moraru et al, 2003). Based on these two
approaches, synthesis of NPs or nanomaterials can be broadly classified into two main
categories: 1. Physical synthesis methods and 2. Chemical synthesis methods. Several
physical and chemical synthesis methods have been reported to design, fabricate, and
manipulate nanostructured materials by innovative approaches (Hu et al, 2009). In
32
addition, several reviews and book chapters are available on the synthesis and properties
of different types of NPs such as metal NPs, semiconductor NPs, carbon-based NPs, and
NPs in general. Burda et al (2005) provided a more comprehensive list of methods to
synthesize NPs. Chen and Mao (2007) as well as Linsebigler et al (1995) reported various
approaches of TiO2 NP synthesis, their properties, modifications and applications.
Nanostructured TiO2 materials in the form of NPs, nanorods, nanowires, nanotubes,
films or coatings, and nanoporous structures can be prepared by using various physical
and chemical synthesis methods. Physical synthesis methods are based on subdivision of
bulk materials (top-down approach) and the NPs thus produced are usually large in size
and have wide size distribution. Most commonly used physical synthesis methods include
but not limited to ball milling, thermal decomposition, laser ablation, arc-ion plating,
spray pyrolysis, flame pyrolysis, magnetron sputtering, ion-beam sputtering, and physical
vapor deposition etc. On the other hand, chemical synthesis methods are based on the
reduction of metal ions or decomposition of precursor solutions to form atoms followed
by aggregation into nano-sized particles (bottom-up approach). The NPs thus prepared by
chemical synthesis methods usually have a narrow size distribution and good control over
composition. Sol-gel synthesis, hydrothermal treatment, sonochemical method, co-
precipitation, anodic oxidation, and electrophoretic deposition are the most common
examples of chemical synthesis methods.
2. Methods to develop antimicrobial nanocoatings
Goddard (2011) stated that the antimicrobial agents commonly used for coating
on food contact and non-food contact surfaces can be classified into two categories. 1.
Migratory and 2. Non-migratory antimicrobial agents. Migratory antimicrobial agents are
33
blended throughout the bulk of the material. These antimicrobials exhibit activity through
migratory effect and do not need close contact of bacterial cell to the antimicrobial agent.
However, their antimicrobial activity diminishes over a time, limiting its long time
functionality (Goddard, 2011; Page et al, 2009). In contrast, non-migratory antimicrobial
agents are strongly attached to the food processing surface by the strongest possible
chemical bond and therefore less likely to migrate from the surface (Goddard, 2011).
Such non-migratory antimicrobial materials therefore have the potential for long-lasting
antimicrobial activity and have the added benefit of being unlikely to migrate to the food
product. Further, non-migratory antimicrobial materials require direct contact with
microorganisms to be effective (Goddard, 2011).
Several methods have been proposed to develop inorganic and organic nanocoatings
on different materials. Visai et al (2011) presented an overview of available surface
technologies allowing the deposition and/or structuring of TiO2 films with antibacterial
properties. Bastarrachea et al (2015) reported various methods used to develop
antimicrobial coatings on food equipment surfaces. In the following section most
commonly used approaches for coating were briefly summarized largely based on the
above mentioned two review papers.
2.1. Graft polymerization: Graft polymerization is a widely used method for coating
on polymeric substrates. However, steel and other inorganic metals can also undergo
graft polymerization (Bastarrachea et al, 2015). The surface chemistry of a solid support
will be changed by grafting polymeric chains with desirable characteristics (Kato et al,
2003). Two different approaches are followed in graft polymerization (Bastarrachea et al,
2015). In the first approach, the solid surface will be pre-treated with either
34
gamma/UV/high-energy electron beam irradiation or by treatment with ozone, plasma,
corona, or flame to create reactive group in order to initiate grafting of antimicrobial
monomers. In the second approach a preformed polymers are immobilized onto a
functionalized solid support.
2.2. Cross-linkable coatings: Cross-linkable coatings are polymers that can bond
with each other after deposition by inclusion of chemical cross-linkers or subsequent
exposure to irradiation or heat curing. UV curing and chemical treatments are the most
often used methods of cross-linking polymers (Bastarrachea et al, 2015).
2.3. Self-assembled monolayers: Self-assembled monolayers form when molecules
spontaneously form into a single layer of relatively ordered groups on a material surface
via strong interaction between anchoring group and the surface (Bastarrachea et al, 2015).
N-alkyl silanes on hydroxylated inorganic surfaces such as silica, glass, steel and thiols
on gold are some common examples of self-assembled monolayers (Raynor et al, 2009).
Self-assembled monolayers have a thickness of one to several nanometers and relatively
simple to deposit over large surface areas. However, their stability under aqueous
conditions has not been well established and remains challenge to commercial adaptation
(Bastarrachea et al, 2015).
2.4. Langmuir-Blodgett films: Langmuir-Blodgett films are comprised of single or
multiple layers of highly organized surfactants that form when a solid support is removed
from a solution containing the material to be deposited (Zasadzinski et al, 1994). Unlike
self-assembled monolayers, these films are rely on physisorption rather than
chemisorption and covalent bond formation and are therefore not as stable as self-
assembled monolayers.
35
2.5. Layer-by-Layer: Layer-by-Layer (LbL) self-assembly is a method by which a
multiple layer coating/film of nanometer-thick layers can be made by sequential
adsorption of oppositely charged polyelectrolytes on a solid support (Bastarrachea et al,
2015). Various antimicrobial components can be incorporated into the bilayers, such as
silver NPs (Dubas et al, 2006), N-halamines (Bastarrachea and Goddard 2013),
quaternary ammonium compounds (Grunlan et al, 2005), and chitosans (Gomes et al,
2013). LbL assembly is rapid, cost-effective, conformal coating technique that can create
durable layers on many surfaces, from polymers to stainless steel (Bastarrachea et al,
2015).
2.6. Electroplating or Electroless plating: Electroplating is a solution-based process
that creates a thin coating of metal on another metal by applying an electrical current. In
contrast, electroless plating is an autocatalytic plating method that uses a chemical
reducing agent in the bath instead of electricity (Mallory and Hajdu, 1990). The thickness
of electroless-plated coatings is typically limited to a few micrometers, and thorough
cleaning of the surface to be plated is critical for optimal adhesion (Ghodssi and Lin
2011).
2.7. Electrophoretic deposition: Electrophoretic deposition is a technique that
exploits the movement of charged particles in suspension in the presence of an
appropriate electric field. In electroplating the coatings are built from metallic ions
converted into atoms when discharged at cathode, whereas in electrophoretic process the
coatings are formed by a deposition of relatively large powder particles. Electrophoretic
deposition allows the deposition of coating from almost any material class, including
metals, polymers, and ceramics. (Visai et al, 2011).
36
2.8. Anodic oxidation: In this method an electrical field driven metal and oxygen ion
diffusion lead to formation of an oxide film at the anodic surface. The anodic oxide film
growth is a two-stage process that results in either a thin or thick TiO2 film. A linear
growth in the nanometric range of the TiO2 film is achieved up to 160 V of applied
voltage drop in the electro chemical cell. However, when anodization carried out at
higher voltages, an increased gas evolution and often sparkling are obtained, resulting in
TiO2 films of higher thickness. This process is generally called micro-arc oxidation or
anodic spark deposition. Doping of metal ions in TiO2 films is also possible with anodic
oxidation process. The films show controlled porosity, morphology, chemical
composition, and allotropic structure (Visai et al, 2011).
2.9. Chemical vapor deposition: In chemical vapor deposition (CVD), a thin film is
formed on a heated solid support from a gaseous phase in a closed chamber, followed by
removal of unreacted gas and chemical by-products from the chamber. A commercial
limitation to CVD coating of food processing equipment is that the apparatus required for
CVD processing is complex, and the size and throughput of the materials to be coated is
limited. Nevertheless, the robust, high purity coatings imparted by CVD may be useful in
certain applications in food processing equipment such as small and irregularly shaped
sanitary valve components (Bastarrachea et al, 2015). Sobczyk-Guzenda et al (2013)
found that the radiofrequency plasma enhanced chemical vapor deposited TiO2 coatings
has exhibited better mechanical properties and bactericidal activity compared to sol-gel
synthesized TiO2 films.
2.10. Plasma spraying: Plasma spray is also a widely used technique to form ceramic
and oxide coatings on wide range of inorganic substrates. The process is based on the
37
action of an electric arc that melts and sprays materials onto a solid surface. Generally,
the material to be deposited is injected in powder form using an inert gas such as argon as
powder carrier. Ctibor et al (2012) created TiO2 powder coatings on stainless steel
substrate using water stabilized plasma gun with argon or nitrogen as carrier gases. They
found that argon assisted TiO2 coatings found to be slightly more stable than nitrogen
assisted TiO2 coatings. However, nitrogen assisted coatings were found to be
photocatalytically more active in decomposing acetone.
2.11. Sol-gel synthesis: Sol-gel synthesis method is a wet chemical technology based
on a sequence of synthesis steps. This method involves hydrolysis and condensation of
metalloorganic alkoxide precursors. Sol-gel technique allows the possibility of
incorporating metal ions, nanometric clusters, and bactericidal molecules (Visai et al,
2011). Sol-gel coatings can be deposited mainly either via dip, spin, or spray coating.
This technique is particularly useful in producing advanced antibacterial coatings due to
the simple industrial scale-up and the esthetic quality of the resulting film. In addition,
sol-gel synthesis is the most widely reported method in the literature. A typical sol-gel
process consists of three major steps:
i) Synthesis of NPs
ii) Coating
iii) Heat treatment
i) Synthesis of NPs
In the first step of sol-gel synthesis, a colloidal suspension, or a sol, is formed
from the hydrolysis and polymerization reactions of the precursor solutions. Complete
polymerization and subsequent loss of solvent leads to the transition from the liquid
38
solution (sol) into solid (gel) phase (Chen et al, 2007). TiO2 NP synthesis and coating by
sol-gel approach is the most popular technique used in several studies. Different types of
precursor solutions, organic solvents along with various surfactants, stabilizers, and
binding agents will be used for this purpose. Process conditions during hydrolysis and
polymerization reactions in sol-gel synthesis are crucial to achieve NPs of desired
characteristics. Sugimoto et al (2003) conducted extensive studies on sol-gel method for
the formation of TiO2 NPs of different sizes and shapes by tuning the reaction mixtures
and by use of different shape controllers. Oskam et al (2003) reported the time-
temperature dependence of NP formation during sol-gel synthesis. They found that the
rate constant for coarsening increases with temperature due to temperature dependence of
the viscosity of the solution and the equilibrium solubility of TiO2. They also reported the
formation of secondary particles at longer times and higher temperatures. In another
study by Barati et al (2009) investigated the effect of pH on homogeneity and particle
size of the precursor solution. They found that the TiO2 crystal size decreases by
decreasing the pH of precursor solution.
ii) Coating
In the second stage, the synthesized sol-gel suspension was coated on a substrate
using different approaches. Dip coating and spin coating are the most widely reported
approaches in the literature. In dip coating method the substrate will be dipped in a
precursor solution for a specified time and later slowly pulled out from the precursor
solution at a specified withdrawal speed. As the substrate pulled up, the film dries due to
convectional air movement. Later, it will be subjected to heat treatment in an oven at
higher temperatures for a specified duration. By repeating this process a thin film of
39
desired thickness will be formed on the substrate. Whereas in spin coating, the substrate
will be mounted on a spin coater and the precursor solution of certain volume will be
poured on the surface. Later, the substrate will be rotated at a certain speed (rpm) for a
specific period of time. The centrifugal force generated during this process spreads the
sol as a thin film on the substrate by expelling excess solution outside. After drying the
film as described before, the process will be repeated to generate a film of desired
thickness.
The above methods are the most simple and efficient. However, as such they are
not practically feasible methods of application in commercial scale and have some
limitations. Alternatively, different coating techniques were used in several studies.
Marcos et al (2008) developed TiO2 coatings on glazed ceramic tiles using screen-
printing method. Anatase TiO2 powder was printed through different sieved screens (55
and 136 µm) on glazed tiles. MacFarlane et al (2011) developed physically stable and
robust TiO2 surfaces on aluminum substrates by using jet spray screen printing method
adopted from Marcos et al (2008). Cerna et al (2011) followed a novel method of coating
using an inkjet printer. In this method the prepared TiO2 solution was deposited on glass
substrate of different film thickness by adjusting printer cartridge and print settings.
Parthasarathy et al (2009) used a hand held spray gun for coating TiO2 on textile fabrics.
A dispersion of NP was filled in a hand spray gun and the fabric substrate was fixed on a
vertical board. The suspension was evenly sprayed over the fabric by maintaining a
constant distance between the fabric and spray gun nozzle. In another study by Ricon et
al (2001) used a spray gun to paint the TiO2 emulsion on to stainless steel substrates.
Film thickness was controlled by the number of spray cycles. The thickness of the 15-
40
cycle spray-painted coating, after sintering at 450°C in air, is roughly around 5-6 µm.
Shinde et al (2008) used a pneumatic spray system to coat TiO2 thin film on glass. The
coating parameters were optimized to obtain uniform, homogeneous and adherent thin
films up to 800 nm thickness. Witanachchi et al (2006) used laser-assisted spray pyrolysis
for the growth of TiO2 and Fe2O3 NP coatings on silicone substrates. Coatings with much
smaller and well-defined grains have been grown by laser heating the droplets. Tomeszek
et al (2006) reported plasma sprayed TiO2 functional coating on SS substrate with a
thickness range of 30 to 50 µm. Structural characterization of these films revealed better
properties than powder deposition techniques. Taniguchi et al (2003) reported successful
fabrication of La1-x Srx Co1-y Fey O3 thin films by electro static spray deposition and
studied the effect of various deposition temperatures, deposition times and liquid flow
rates on the film structure. Ctibor et al (2012) analyzed structural, mechanical and
photocatalytic activities of TiO2 thin films created by using water-stabilized plasma gun.
iii) Heat treatment
In the third step of sol-gel synthesis, the coated substrates will be first preheated
to evaporate the solvent followed by calcination or sintering at higher temperatures to
create stable thin films. Several studies have reported that drying at around 100°C for 30-
60 min followed by calcination in the range of 400 to 600°C is ideal for obtaining anatase
form of crystalline TiO2 thin films with good photocatalytic activity. Mathews et al
(2009) studied the effect of annealing temperature on structural, optical and
photocatalytic properties of TiO2 thin films on soda lime glass slides. The dip coated
films were dried in air at room temperature and later heated for 2 min in oxygen at
100°C. Subsequently, the coatings were calcinated at 200-600°C under oxygen flow.
41
Their results showed that the structure of the film changes from amorphous to
polycrystalline after annealing at 400°C and the band gap of the created film decreased
from 3.4 to 3.32 eV after annealing at 600°C. Barati et al (2009) reported that the TiO2
thin films created on stainless steel showed anatase structures when calcinated at 350-
550°C followed by solvent bath drying. Vigil et al (2009), used microwave assisted
chemical bath for deposition of TiO2 thin films on stainless steel using different precursor
solutions. The thin film coated coupons were microwave irradiated (2.45 x 109 Hz. 0.6
kW) and subsequently calcinated at temperatures lower than 700°C. Results showed that
amorphous TiO2 was deposited on stainless steel from the TiOSO4 precursor solution
while a disordered anatase phase was deposited from the (NH4)2TiF6 precursor solution.
Ilmenite (FeTiO3), as well as, hematite (Fe2O3) appeared with heat treatment, indicating
that Fe ions diffuse into the TiO2 film. Yang et al (2007) subjected the dip coated TiO2
thin film to heating for 5 min using a domestic microwave oven. The resultant thin film is
well characterized with smooth morphology and spherical shape with grain size of 68.2
nm and strong absorption band in the range of 300-387 nm with band gap energy of 3.4
eV. Alrousan et al (2009) dried TiO2 thin films using IR lamp in between the coats.
Machida et al (2005) studied the antibacterial efficacy of TiO2 film photo-deposited silver
ions on tiles which are first air-dried and later calcinated at 880-980ºC for 1 hr. Results
showed that when calcination temperature was < 900ºC, the antibacterial activity was
100%, irrespective of TiO2 thickness. They also found that the calcination temperature,
film thickness has significant impact on anatase to rutile composition, and photo
deposited silver.
42
The above mentioned studies indicate that the synthesis of NPs beginning with a
precursor solution is a complex process and need to control several variables to obtain
desired characteristics. In addition, aggregations of NPs, crack formation on films are
some common problems that are widely reported in the literature. Alternatively, few
studies reported the use of commercially available ready-made TiO2 NPs dispersed in an
organic solvent to form sol-gel for direct coating. This method of approach limits the
complexity of choosing suitable chemical ingredients for precursor solutions and control
over the reaction parameters. Marcos et al (2008) suspended TiO2 powder with 0.29 µm
size and 12.94 cm2/g specific surface area in an organic media and the resultant
suspension was used for coating on glazed ceramic tiles. Similar technique was used by
MacFarlane et al (2011) on aluminum substrates and reported the formation of uniform
coatings with high surface area and physical stability. Kim et al (2008) used aqueous
solution of commercial Degussa P25 powder mixed with carbowax binder (PEG) to
deposit thin films on glass substrate and tested their antimicrobial activity. Their results
showed that number of coatings on glass showed no difference in the antimicrobial
activity. Similarly, Alrousan et al (2009) used a suspension of Degussa P25 in methanol
to develop TiO2 thin films for bacterial inactivation in surface water. Machida et al
(2005) sprayed commercial TiO2 solution on ceramic tiles (3 g/m2) and studied the
antibacterial efficacy of resultant TiO2 thin film.
2.12. Wet chemical methods using different binding agents: Wet chemical methods
involve use of an organic/inorganic binder and a suitable solvent along with an
antimicrobial agent in order to prepare a suspension for coating on a substrate. Depending
upon the nature of the constituents used in the coating, a subsequent heat treatment may
43
be required to evaporate the solvent and to remove the organic binder from the coating to
achieve high bactericidal property and physical stability. Three different mechanisms
namely oxidation, solvent evaporation, and polymerization or chemical cross-linking will
be involved while creating coatings using a binding agent. Oxidation of binder in the
coating lead to thermosets which remain hard on exposure to heat. Usually this type of
coatings contains drying oils which take time to dry and achieve good moisture as well as
chemical resistance. However, over a time these coatings lead to cracking, embrittlement,
and deterioration. Solvent evaporation of coatings leads to thermoplastics which deform
or soften by exposure to heat. In general, water or organic solvents are used in these types
of coatings. Whereas, polymerization or chemical cross-linking mechanism leads to
thermosets.
Many studies have reported the use of organic and inorganic binding agents for
developing nanocoatings on different substrates. Kasanen et al (2011) studied the UV
stability of polyurethane binding agent on multilayer photocatalytic TiO2 coating on glass
substrate. They found that the optimal dilution of polyurethane binder in water is 1:4 for
better photocatalytic activity and binding of TiO2 to the substrate. Similarly, Bhargava et
al (2012) investigated the effect of TiO2 concentration (pigment-to-binder ratio) and
dispersing agent percentage on the peel strength of high reflectivity waterborne
polyurethane based coatings on aluminum substrate. Dhoke et al (2012) developed
polyurethane based ZnO coatings on stainless steel. Their results showed 0.1% ZnO with
polyurethane showed better UV, scratch, and abrasion resistance. Qiao et al (2012)
fabricated boron carbide green sheets using a suspension with polyvinyl butyral as a
binding agent, di-n-butyl phthalate as a plasticizer, castor oil as dispersant and single
44
oleic acid glycerol as wetting agent. Their study found that shear thinning behavior of the
slurry was found to be the most suitable for coating. In addition, optimal contents of
binder and plasticizer were found to be 5 wt %, wetting and dispersing agents were found
to be 2 wt%. Addition of diethylene glycol (DEG) while preparing TiO2 precursor
solution using titanium isopropoxide and ethanol enhanced the thin film physical
characteristics such as adherence, robustness, and surface smoothness on stainless steel
surface (Kajitvichyanukul et al, 2005). Also, the photocatalytic activity of resultant TiO2
coating remained unchanged when compared with the control. Similar results were also
observed by Cerna et al (2010) when using polyethylene glycol (PEG) in precursor
solution for thin film formation on glass substrate. In another study by Fretwell et al
(2001) added PEG and DEG in a precursor solution along with ethanol as solvent to
avoid aggregation of titania particles. Addition of PEG and DEG helped to form a
network with NPs and avoid aggregation. Tsoukleris et al (2007) and Chorianopoulos et
al (2011) prepared TiO2 pastes by using different solvents, rheology agents, and surface
modifiers along with PEG for coating on glass and stainless steel substrates. In addition,
Chorianopoulous et al (2011) tested TiO2 coated glass and steel substrates prepared with
Triton X-100 surfactant to disinfect Listeria monocytogenes biofilms in a food processing
environment.
Shi et al (2008) used an epoxy resin (Bisphenol-A) based coating embedded with
TiO2 NPs. Their study found that epoxy resin showed good chemical (acid/alkali), heat,
and wear resistance but has high rigidity, weak photostability due to oxidation of
methylene chains. Cheema et al (2012) fabricated optically transparent nanocomposites
with enhanced mechanical properties using stable dispersions of ZrO2 NPs mixed with a
45
commercially available bisphenol-A-based epoxy resin and cured with a mixture of two
amine-based curing agents. Wagner et al (1998) developed novel corrosion resistant hard-
coatings for metal surfaces. A sol-gel precursor solution is prepared by adding inorganic
fillers such as GPTS (ɤ- glycidyloxi propyl trimethoxy- silane) and PTMS (propyl tri-
methoxy-silane). They found that the epoxy group of GPTS generates a polyethylene
oxide network beside the inorganic backbone by organic polymerization. PTMS has been
used as a network modifier to increase the relaxation behavior of the material to achieve a
more hydrophobic behavior of the material. Bhave (2007) reported a good adhesion of
GPTS and MTMS based TiO2 (brookite phase) coatings on glass substrate when
compared to coating consisting of Degussa P 25 TiO2. The differences in the adhesion
strength of TiO2 coatings might be attributed to the poor wetting and dispersion
properties of Degussa P 25 compared brookite phase TiO2 NPs. Schmidt et al (1997)
created a corrosion resistant nanocomposite coating using GPTS, and SiO2 NPs by wet
coating method. These coatings showed excellent abrasion resistance and the physical
stability of these coatings was found to be in the order of anodizing processes. Dhoke et
al (2009) created ZnO nanocoatings on stainless steel substrates using waterborne
silicone modified alkyd resin, a cross-linking agent and a neutralizing medium. They
found that by increasing the concentration of ZnO, scratch resistance and abrasion
resistance of the coatings increased. Similarly, Allen et al (2004) used TiO2 NPs to mix
with alkyd paint for coating on stainless steel substrates. Faure et al (2011) used silicone
co-polymers along with an antimicrobial agent nisin for dip coating on stainless steel.
They found that the coatings are durable and exhibited good microbicidal activity. Li et al
46
(2009) created ZnO coating on plastics using acrylic resin mixed with a curing agent and
a thinner.
Gergely et al (2011) developed corrosion resistant zinc rich alumina coatings on
stainless steel surface using a two component mixture consisting of epoxy resin and a
cross-linking agent. Caballero et al (2010) suspended TiO2 NPs in acrylic paints along
with extenders such as CaCO3, silica, and talc at different TiO2 concentrations. The paint
formulations were then coated on polyester sheets and tested for photocatalytic
bactericidal activity. Kumar et al (2012) mixed TiO2 NPs that are functionalized with
silanes with epoxy paint and cured with cycloaliphatic amine. The suspensions were then
spray coated on carbon steel. They found that scratch hardness, adhesion strength,
abrasion resistance, flexibility, and corrosion resistance were improved with silane
treated TiO2 in the coating when compared to untreated TiO2. Marolt et al (2011) studied
the photocatalytic activity of TiO2 coating on card board using acrylic binder.
Han et al (2012) prepared TiO2 coated polyester fibers using colloidal silica as binding
agent. They reported that the spray-coated samples of TiO2 showed higher photocatalytic
activity and physical stability when compared to dip coated samples.
3. Methods to evaluate surface characteristics and physical stability of
nanocoatings
Understanding the structural characteristics NPs and nanocoatings such as size,
shape, chemical composition, and orientation etc, are important for estimating their
photocatalytic properties. Several techniques such as X-ray diffraction (XRD), X-ray
photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), transmission
electron microscopy (TEM), atomic force microscopy (AFM), optical, and Raman
47
spectroscopy were widely used techniques for this purpose. In addition, evaluating the
physical stability of the nanocoatings is vital for estimating their long-term durability.
Several methods can be used for this purpose. Scratch hardness of the coating can be
determined based on ASTM G171-03 standard method. This method characterizes the
resistance of a solid surface to penetration by a moving stylus of given tip radius under a
constant normal force and speed. The hardness of coated surface can be expressed as
scratch hardness number in GPa. Adhesion strength of the coating on the substrate can be
evaluated by ASTM D3359-02 method. In addition, ASTM D4060-14 is the standard test
method for measuring abrasion resistance of a coating which is also referred as the Taber
test. The test specimen is mounted to the Taber abrader and rotated at a fixed speed under
a weighted CS-10 or CS-17 abrading wheel. The weight loss per specified number of
revolutions under specified load is expressed as wear index. Simunkova et al (2003)
discussed the tests used for evaluating the mechanical properties such as adhesive
strength, cohesive behavior, wear resistance, micro hardness and fracture hardness of thin
film substrate systems. A reciprocating test to simulate food processing cleaning
operations was mentioned in a report by European Commission which evaluated the
effect cleaning on the antibacterial activity of coated surfaces. In addition, prolonged
water exposure (one week immersion), thermal cycling (5 cycles: 2 min 70°C water + 30
min -18°C), acid (pH 3.1) and caustic (pH 10) exposure (1 hour immersion), manual
brush cleaning and sand falling test were also discussed to measure the physical stability
and antimicrobial performance of the coatings.
48
IV. ANTIMICROBIAL ACTIVITY OF TIO2 NANOMATERIALS
1. Bactericidal activity of TiO2 in suspension vs coating
Since the early work of Matasunga et al (1985), many research groups have
reported the application of semiconductor photocatalysis for the inactivation of different
types of pathogenic microorganisms, such as bacteria, viruses, algae, fungi or protozoa.
As described earlier, the bactericidal properties of TiO2 are attributed to the high redox
potential of the surface species formed by photo-excitation. The type and the source of
TiO2 has shown to exhibit significant effect of bactericidal activity. This is mainly
attributed to the rate of formation of ROS depends on the particle size, crystalline phase,
the isoelectric point, and the specific surface area of the NPs. All these surface properties
can be controlled by the method of synthesis and various with the type of NPs. On the
other hand, the biological parameters of the microorganisms such as microbial species,
growth phase, initial cell density etc., were also found to have significant effect of
photocatalytic disinfection. In addition, the experimental conditions such as the
concentration of NPs, the light intensity, the wavelengths, and the treatment time were
also important (Hitkova et al, 2012). Most recently, our studies on TiO2 bactericidal
activity in suspension revealed that the type and source of NPs, bacterial cell harvesting
conditions, volume of reaction mixture, and the intensity of UV light has showed
significant effect on the bactericidal property of TiO2 NPs (Yemmireddy and Hung,
2015).
Hitkova et al (2012) studied the antibacterial activity of sol-gel synthesized TiO2
NPs in suspension at 1 mg/mL concentration. Their results showed that E.coli were more
susceptible to photocatalytic disinfection when compared to P. aeruginosa, and S.
49
aureus. Faure et al (2011) studied the photocatalytic inactivation of TiO2 on five different
photocatalytic supports, in terms of TiO2 type and source (Degussa P25 vs Millennium
PC500) and configurations (catalyst was impregnated on supports, alone or with binder,
or suspended in water). They found that for the same type of TiO2, inactivation efficiency
was better in suspension (up to 4 log in 2 h) followed by TiO2 impregnated without
binder (up to 2 log in 2 h) and finally TiO2 with binder (only 0.5 log after 2 h). Although,
several studies have found that TiO2 in suspension has high microbicidal property, its
practical application in suspension is limited due to difficulties in post-reaction catalyst
separation. Many efforts have been devoted to immobilize NPs on inert supports for more
practical applications.
Kuhn et al (2003) studied the photocatalytic bactericidal activity TiO2 coatings on
glass substrate using different bacterial strains. They reported that the order of bacterial
susceptibility to photocataytic disinfection is as follows: E.coli > P. aeruginosa > S.
aureus > E. faecium > C. albicans. Marugan et al (2008) found that TiO2 supported on
silica show a significant decrease in the bactericidal activity when compared with
bacterial activity of TiO2 (Degussa P-25 ) in suspension. They also reported that in a
coated supports, the contact between TiO2 and the microorganisms is limited to the TiO2
crystal located in the external surface of the particles. This area represents only a small
fraction of the semiconductor loading that is available for actual photo-killing effect.
Chorianopoulos et al (2011) developed TiO2 (Degussa P-25) coatings on stainless steel
and glass substrates using acetyl acetone as a solvent and Triton X-100. These coatings
reduced Listeria monocytogenes biofilms by 3 log CFU/cm2 after 90 min UVA light
illumination. Kim et al (2008) created TiO2 thin films on glass substrate by using
50
Degussa P-25 and carbowax binder. They reported that the number of coatings had no
effect on the antimicrobial property. Evans et al (2007) developed TiO2 thin films on the
surface of stainless steel using a combination of flame-assisted chemical vapor deposition
(FACVD) and thermal atmospheric pressure –assisted chemical vapor deposition
(APCVD). They reported a 6 log reduction of E.coli in less than 3 h when irradiated the
TiO2 coatings at 2.2 mW/cm2 UVA light intensity. However, the intensity of UV light
alone used in this study is high enough to show significant bactericidal activity. In
another study by Ditta et al (2008) reported the antibacterial activity of APCVD coated
TiO2, CuO, and TiO2/CuO dual layers on E. coli and bacteriophage T4. Their results
suggested that the bacteriophage T4 was more susceptible to photocatalysis than E.coli.
Among the tested coatings, TiO2/CuO composite coatings showed higher efficacy which
might be attributed to the additional toxicity of Cu ions. George et al (2010) studied the
bactericidal performance of flame-sprayed TiO2 and TiO2-Cu composite coatings against
Pseudomonas aeruginosa. Under the same conditions, the TiO2-Cu composite coatings
had the same bactericidal capability as pure Cu surfaces. The change of TiO2 from
anatase to rutile phase during high temperature flame spraying was found to be one
possible reason for the low bactericidal property. However, the composite coatings
showed improved bactericidal performance under light irradiation. MacFarlane et al
(2011) studied the photocatalytic microbicidal activity of jet spray formed TiO2 surfaces.
The microbial inactivation rates were highest for P. aeruginosa (Gram-negative)
followed by S. aureus (Gram-positive) and C.albicans (yeast). Xiao et al (2014) reported
antibacterial and antifungal activity of Fe-doped TiO2 coating with chitosan under visible
light irradiation. A slurry of Fe- doped TiO2 mixed with chitosan using a crosslinking
51
agent (epichlorohydrin) was spread coated on glass slides. They reported that the
composite coatings were very efficient in reducing the levels of the three tested strains
E.coli, C. albicans, and A. niger.
2. Studies related to bactericidal activity of TiO2 for food safety applications
TiO2 has been approved by the American Food and Drug Administration (FDA)
for use in human food, drugs, cosmetics, and food contact materials (FDA, 2014) and it
permits up to 1% of TiO2 as an inactive ingredient in food products. Also, TiO2 has been
evaluated positively as a food additive by European Union (EU Directive 94/36/EC,
1994). It is widely used as an additive in various products such as cosmetics (e.g.
lipsticks), food (e.g. salami, chewing gum, cookies) and pharmaceuticals as a white
pigment. Malato et al (2009) reported that TiO2 is the most active photocatalyst under the
photon energy of 300 nm< l < 390 nm and remains stable after the repeated catalytic
cycles. TiO2 can kill both Gram-negative and Gram-positive bacteria, although Gram-
positive bacteria are less sensitive due to their ability to form spores (Wei et al, 1994).
However, contrasting information is available regarding sensitivity of Gram-negative and
Gram-positive bacteria towards TiO2 photocatalysis.
TiO2 photocatalytic activity has been found particularly useful to decontaminate
wash water used for cleaning minimally processed products (Chaleshtori et al, 2008).
TiO2 coatings on food contact surfaces was found to diminish the growth of Listeria
biofilms (Chorianopoulos et al, 2011) or to improve cleanability of stainless steel (Verran
et al, 2010). The biocide capacity of TiO2 nanocomposites with typical packaging
materials has also been tested (Cerrada et al, 2008; Diaz-Visurraga et al, 2010). Kim et al
(2009) reported a reduction of up to 2.8 log CFU/g for E.coli, L. monocytogenes, S.
52
aureus and S. typhimurium in inoculated iceberg lettuce when germicidal UV light (254
nm) illuminated through a TiO2 coated quartz glass. In vitro studies using TiO2 coated
polypropylene films were effective in decreasing the counts of E. coli up to 3 log CFU/g
(Chawengkiwanich and Hayata, 2008). In the same study, a reduction of over 1 log
CFU/g was observed during extended storage. Their results also showed that the
antimicrobial effect of TiO2 coated film is dependent on the UVA light intensity, but it is
independent of the particle size of TiO2 used for coating. Similarly, Maneerat and Hayata
(2006) reported antifungal activity of TiO2 powder and TiO2 coated plastic film to
prevent Pencillium fruit rot on apples, tomatoes, and lemons. Their study revealed that
the concentration of TiO2 and the growth of Penicillium expansum were inversely
correlated. Chorianopoulos et al (2011) reported photocatalytic disinfection potential of
TiO2 coatings on stainless steel and glass substrates against Listeria monocytogens
biofilms. Wang et al (2010) found that a nano-packaging containing Ag and TiO2 in
combination with a hot air treatment were efficient in improving green mold control and
ethylene production in Chinese bayberries. TiO2 photocatalysis has also shown to be
effective for the inactivation of foodborne pathogens such as Salmonellas spp, Vibrio
parahaemolyticus, and Listeria monocytogenes (Kim et al, 2008). Bodaghi et al (2013)
developed TiO2 nanocomposite packaging film through melt blending technique using an
extruder. They reported a 4 log and 2 log CFU/mL reduction of Pseudomonas spp and
Rhodotorula mucilaginosa, respectively when using these packaging films. Further, in
vivo tests on fresh pears packaged in TiO2 nanocomposite film showed significant
reduction in mesophilic bacteria and yeast growth when stored at 5 °C for 17 days.
53
Studies with E.coli strains (PHL 1273) synthesizing curli, a type of appendage
that allows the bacteria to stick to surfaces and form biofilms, found that titania and
various types of UV irradiation were able to inactivate this organism. In dark event
studies, following the bacterial inactivation, no bacterial cultivability was recovered even
after 48 h, indicating that the durability of the TiO2 disinfection was adequate (Gamage et
al, 2010). In a study by Gelover et al (2006), found that a complete inactivation of fecal
coliforms was achieved in 15 min by exposing water in TiO2 coated plastic containers to
sunlight whereas the same extent of inactivation required 60 min with uncoated
containers. This study also found that the bacteria exposed to TiO2 photocatalytic
disinfection do not self-repair. Nano-sized TiO2 was also reported to kill viruses
including poliovirus-1 (Watts et al, 1995), hepatitis B virus (Zan et al, 2007), herpes
simplex virus (Hajkova et al, 2007), and MS2 bacteriophage (Cho et al, 2005). Nakano et
al (2012) reported antiviral properties of TiO2 thin film coatings against influenza virus.
This study also investigated the effect of UV intensity, irradiation time, and bovine serum
albumin concentration in the viral suspensions on the inactivation kinetics.
3. Considerations for testing antimicrobial activity of photocatalytic nanomaterials
Several studies in the literature have used different approaches to determine the
antimicrobial property of photocatalytic nanocoatings. However, the International
Standard Organization (ISO) suggested a standard method ISO 27447 for evaluating
antibacterial activity of semiconducting photocatalytic materials (ISO, 2009). This
standard specifically applies to evaluate antibacterial activity on photocatalytic ceramic
materials and other materials that are generated through coating or mixing with
photocatalysts. Similarly, ISO 13125 (E) is the standard method for testing antifungal
54
activity of semiconducting photocatalytic materials (ISO, 2013). Though each of these
methods including the standardized methods that are followed in the literature have their
own advantages and limitations, the variety of methods used to determine antimicrobial
activity impedes an accurate comparison of the test results.
The approaches that are used to test the antimicrobial activity of photocatalytic
nanomaterials based on the method of inoculation can be broadly classified into:
i) Direct inoculation method
ii) Glass cover-slip method
iii) Direct immersion method
iv) Indented well method
v) Agar diffusion method
3.1. Direct inoculation method
Direct inoculation method is the most widely reported test method in the literature
(Xiao et al, 2014; Faure et al, 2011; Ditta et al, 2008; Evan et al 2007; Kuhn et al, 2003;
Sunada et al, 2003; Yu et al, 2003; Kikuchi et al, 1997). Briefly, in this method a
specified volume test inoculum is directly pipetted on to the surface of photocatalytic
material to be tested. After the treatment, the samples are enumerated as per standard
microbiological recovery protocols. Even though this method is simple to conduct, the
main disadvantages include: (i) non-uniform coverage of the inoculum on the test
surface, and ii) drying out of the inoculum during the photo-treatment. These conditions
may lead to inaccurate determination of photocatalytic antimicrobial property of test
surface.
55
3.2. Glass cover-slip method
Glass cover-slip method is mainly based on ISO 27447:2009 standard test
method. As per this method an adhesion film or glass cover-slip is placed on top of the
inoculum in order to ensure good contact of the bacterial cells to the photocatalytic test
surface. Also, Kim et al (2008) has suggested using polypropylene adhesion film based
on better light transmittance through the polypropylene film. Later, in a review conducted
by Mills et al (2012) outlined the pros and cons of using film and glass cover-slip
methods. The main issue associated with using the cover slip method is that it is difficult
to avoid leakage of test inoculum from the sides of the photocatalytic test surface. In
addition, this method of testing inhibits the contact of sufficient atmospheric oxygen with
the NPs to catalyze the process of photocatalytic disinfection.
3.3.Direct immersion method
Krysa et al (2011) compared two different test methods to evaluate bactericidal
property of TiO2 coatings with a modified ISO 27447:2009 method using adhesive glass.
In the first method (50 cm3 method), TiO2 coatings were immersed in 50 mL bacterial
suspension, and in the second method (3 cm3
method) 3 mL of bacterial suspension was
poured into a petridish containing TiO2 coated sample. Their studies concluded that the
50 cm3 method is only useful to test pure TiO2 coatings with strong bacterial property and
the 3 cm3 method is not appropriate for testing. They also found that 3 cm
3 method did
not allow a clear distinction between the inhibition effect of TiO2 and UV light itself and
also it created several unreactive dead zones in the test system. They found that modified
ISO method using adhesive glass is most suitable compared to other two methods.
Similarly, Nakano et al (2012) reported antiviral properties of TiO2 thin film coatings
56
against influenza virus. Their study also suggested a minor modification of the ISO test
method for antibacterial effects of TiO2 photocatalysis to evaluate antiviral activity.
3.4. Indented well method
The indented well method is not a widely reported approach to test microbicidal
activity of photocatalytic materials in the literature. However, this method has several
advantages in solving some issues discussed in previous methods. Cushnie et al (2010)
used an aseptically sealed glass cell ring on the surface of photocatalytic glass slides.
Later, a 300 µL bacterial suspension was carefully pipetted into the ring cells and the
material subsequently irradiated. Another study by George et al (2010) used pyrex wells
mounted on thermal sprayed TiO2 coatings on stainless steel using a silicone rubber in
order to contain bacterial solutions on to the coated substrate. Similarly, Bonetta et al
(2013) used a glass ring fixed with inert cement on TiO2 coated ceramic tiles to inoculate
bacterial culture and determine the photocatalytic bactericidal property. The advantage of
this type of approach is that it allows the bacterial suspension to be accurately deployed
to a known area of the surface under investigation. Also, this method of testing offers the
advantage of uniform coverage and good contact of bacterial cells with the photocatalytic
surface. However, the relative complexity of mounting a well with good seal limits wide
spread application of this test procedure.
3.5. Agar diffusion method
The agar diffusion method is another approach which is widely used to determine
the bactericidal activity of NPs in general. In this method, the NPs is immersed in
bacterial growth media which is already inoculated with test bacterium. After the
treatment, the size of the growth inhibition zone is a measure of bactericidal activity of
57
NPs. For example, Azam et al (2012) compared the antimicrobial activity of metal oxide
NPs such as ZnO, CuO, and Fe2O3 using agar well-diffusion method and minimal
bacterial concentration. They found that ZnO had the highest antibacterial activity
followed by CuO and Fe2O3 when tested against Gram-positive (S. aureus and B.
subtilis) and Gram-negative (P. aeruginosa and E.coli) bacteria. They also found that
Gram –negative bacteria showed more resistance to NPs compared to Gram-positive
bacteria. However, the agar diffusion method is not suitable for photocatalytic NPs such
as TiO2 since these NPs require light penetration and activation in order to exhibit
significant bactericidal property.
Effect of other test conditions
Suspension medium: The chemical nature of the suspension medium was found to have
significant effect on the photo-killing rate of TiO2 NPs. Cushnie et al (2009) reported that
the greatest antibacterial activity was observed when aqueous sodium chloride solution
was used when compared to aqueous tryptone solution. In contrast, Ditta et al (2008)
reported a similar rate of killing by using either saline or water as re-suspension media.
Growth media: Rincon and Pulgarian (2004) reported that the choice of growth media
for enumerating bacterial recovery following photocatalytic treatment is an important
factor that may affect the experimental results. Their study revealed that the non-selective
medium plate count agar showed 1000-times higher response than that of the selective
media CHROM agar following photocatalytic disinfection of E.coli. Similarly, Faure et al
(2011) compared the bacterial re-growth on two different culture media, such as tryptic
soy agar (TSA) and eosin methylene blue (EMB) agar after photocatalytic treatment.
They found that for the same sample, the number of colonies counted on EMB was
58
always lower than on TSA after photo-treatment. They concluded that the lactose
metabolism of bacteria was severely affected due to photocatalytic treatment and hence
showed variable recovery on different media. McCullagh et al (2007) have suggested that
under certain circumstances where it is considered necessary to use a selective media
then it is also important to include a non-selective agar to substantiate and compare the
results.
Incubation time: Many studies have used different incubation times for bacterial
enumeration following photocatalytic treatment with NPs. Cushnie et al (2009) found that
the factors such as osmatic pressure, and incubation time has significant effect on the
outcome of TiO2 photocatalytic disinfection results. Bacteria treated with only UV light
grew more slowly than those treated with TiO2 and UV, often taking in excess of 24 h to
produce visible colonies.
V. SAFETY CONCERNS ON USE OF NPS
1. Toxicity issues
Despite projected benefits, nanotechnology is raising regulatory issues and public
concern regarding safety and environmental effects. Rossi et al (2014) in their review on
scientific basis of nanotechnology, implications for the food sector and future trends
discussed the major issues associated with toxicity of NPs when used in food
applications. The main issues raised by Rossi et al (2014) include:
i. Knowledge gap on how altered physico-chemical properties of NPs may
influence their toxicological properties when ingested into human body via
food.
59
ii. Current toxicity testing approaches appear to be inadequate to detect all the
toxicity aspects of nano-sized materials.
iii. Various dose metrics such as size and other physicochemical parameters of
engineered nanomaterials has to be explored since mass concentration alone is
not sufficient.
iv. Effect of factors such as dissolution rate, agglomeration, aggregation,
adsorption or binding with other food components, and reactions with acid
and digestive enzymes may affect the fate of engineered nanomaterials in the
gut and more understanding on these aspects is needed.
Dutta and Waldman (2012) studied the effect of Si, ZnO, TiO2, and Ag NPs on
the human epithelial cells in a stomach model as well as in a stomach and intestinal
model and measured the uptake, cell death, cell proliferation, and mitochondrial activity.
Their results showed that the ZnO NPs dissolve in acidic medium in the stomach and
showed modest toxicity while TiO2 and silica had almost no cell toxicity; whereas Ag
NPs are found to be very toxic. In another study, Lin and Mustapha (2012) spiked
engineered NPs such as Ag, ZnO, and TiO2 from a commercial source into various food
samples such as wheat flour, yam, corn starch, and pears and used a combination of
techniques, including Scanning Electron Microscopy (SEM), Energy Dispersive
Spectroscopy (EDS), and Inductive Couple Plasma-Optical Emission Spectroscopy (ICP-
OES) to detect the NPs. In addition, they also exposed the NPs to natural gut microflora
such as E.coli, Lactobacillus acidophilus, and Bifidobacterium and human intestinal
epithelial cells to different concentrations of ZnO and Ag NPs. Their results showed that
the tested NPs have some antibacterial properties that inhibit growth of bacteria and
60
higher concentrations of NPs showed toxicity to human epithelial cells. Ammendolia et al
(2014) studied the L. monocytogenes behavior in presence of non-UV-irradiated TiO2
NPs with a special focus on biofilm formation and intestinal cell interaction. Their study
revealed that the TiO2 NPs influenced both production and structural architecture of
listerial biofilm in addition to their interaction with intestinal cells.
The International Agency for Research on Cancer (IARC), after extensive studies
has rated TiO2 nanoparticles as carcinogenic (Group 2b) for humans (Baan et al, 2006).
In vivo studies on the ability of TiO2 NPs to penetrate the GI tract has revealed that the
NPs can be found in systemic organs after an oral exposure of 10 days (Jani et al, 1994).
Liu et al (2010) reported that TiO2 NPs showed intracellular accumulation of ROS
leading to apoptosis in PC12 cells. In addition, Tassinari et al (2013) demonstrated that
reproductive and endocrine effects of short term oral exposure to low doses 0-2 mg/kg
body weight per day of TiO2 NPs. However, due to lack of sufficient data supporting the
toxicity of engineered NPs and the scientific consensus on this matter, further studies
need to be warranted to evaluate potential toxicity of engineered NPs on human health
and the environment.
2. Regulatory framework
For regulatory purposes related to use of nanotechnology in food sector, various
countries have adopted different and sometimes diverging approaches (Cushan et al,
2012). The National Nanotechnology Initiative (NNI) is a U. S. Government’s
multiagency, multidisciplinary research and development program on nanotechnology. It
brings together the expertise of 25 federal agencies and supports collaborative research
and development in academic, government, and industry laboratories (National
61
Nanotechnology Initiative, 2010). The U.S. Food and Drug Administration (FDA) did not
issue strict definitions of NPs and considers food manufacturing processes that involve
nanotechnology in the same manner as any other food manufacturing technology (FDA,
2011). However, the FDA clearly states that, as with any studies to support the safety of
food substances, studies to establish the safety of food substances manufactured using
nanotechnology should have been appropriately validated (FDA, 2012). The Center for
Food Safety and Applied Nutrition (CFSAN) at the U.S. FDA has a research program to
explore the safety assessment of nanomaterials in food and cosmetic products. It focuses
on examining the possibility of NPs leaching from food packaging materials and
determining whether there is a safety concern, and investigating approaches to study the
potential toxicity of NPs (Mermelstein, 2013). Further, the FDA’s National Center for
Toxicological Research is developing analytical tools and procedures to quantify
nanomaterials in complex matrices and conducting toxicity studies on NMs. In addition,
The U.S. National Toxicology Program (NTP) through its Nanotechnology Safety
Initiative, conducting research on several classes of nanoscale materials, including metal
oxides, fluorescent crystalline semiconductors, carbon based fullerenes, carbon
nanotubes, nanoscale silver, and nanoscale gold (Mermelstein, 2013).
The European Food Safety Authority (EFSA), in its scientific opinion on the potential
risks arising from nanoscience and nanotechnologies on food and feed safety, affirmed
that the risk assessment paradigm (i.e. hazard identification, hazard characterization,
exposure assessment and risk characterization) is applicable for engineered nanomaterials
(EFSA, 2009). However, since different types of engineered nanomaterials even with the
same chemical composition may vary as to their toxicological properties, the risk
62
assessment of engineered nanomaterials has to be performed on a case-by-case basis. The
EFSA stated that appropriate data for risk assessment of an engineered nanomaterial in
the food and feed area include comprehensive characterization of the engineered
nanomaterial, information on whether it is likely to be ingested in nanoform, and, if
absorbed, whether it remains in nanoform at absorption or not.
VI. KNOWLEDGE GAP
1. Research scope
Foster et al (2011) reported that about 11,000 publications are available on
photocatalysis and out of which about 800 publications reported photocatalytic
disinfection. Henderson (2011) reported that approximately 2400 heterogeneous
photochemistry papers were published in 2008 and out of which 80 % involved TiO2
based materials. This shows the extent of research activity carried out in the area of TiO2
photocatalysis. Over the last decade, the antimicrobial property of photocatalytic TiO2
NPs has aroused much interest in the food sector. However, our knowledge is very
limited on the use of TiO2 photocatalysts for food safety applications. As reported in the
literature, microbial cross-contamination is a major issue in the entire food chain.
Nanotechnology based intervention strategies using photocatalytic TiO2 NPs has great
potential to help reduce the risk of microbial cross-contamination in the food processing
environment. The potential areas of research in this direction may include but not limited
to:
Developing antimicrobial nanocoatings on food contact and non-food contact
surfaces using TiO2 NPs.
63
Developing testing methods to determine the antimicrobial activity of TiO2
nanomaterials.
Determining the bactericidal efficacy of TiO2 nanomaterials in food processing
environmental conditions.
Determining the durability of the nancoatings for use in food safety applications.
64
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Fig 2.1. Semiconductor photocatalysis
Ref: http://dev.nsta.org/evwebs/1952/photocatalysis.html
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Fig 2.2. TEM of a dispersion of TiO2 Degussa P-25 (1 mg/L) in contact
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CHAPTER 3
SELECTION OF PHOTOCATALYTIC BACTERICIDAL TITANIUM DIOXIDE
(TIO2) NANOPARTICLES FOR FOOD SAFETY APPLICATIONS1
1Veerachandra K. Yemmireddy and Yen-con Hung. LWT- Food Science and Technology.
61 (2015) 1-6. Reprinted here with permission of the publisher.
86
Abstract
The main objective of this study was to develop a systematic testing protocol for
selecting bactericidal TiO2 nanoparticles (NPs). Photocatalytic bactericidal activity of
TiO2 NPs at 1 mg/mL concentration was tested against E.coli O157:H7. The effect of
source of NPs (three different commercial samples referred as T1, T2 and T3), bacterial
cell wash conditions (1 vs 3 wash), volume of reaction mixture (10, 20 and 30 mL) and
intensity of UVA light (1 vs 2 mW/cm2) on bactericidal activity has been determined.
Sample T3 was found to be the most effective among the tested TiO2 samples. Increasing
the number of cell washes from 1 to 3 increased the log reduction (2.91 vs 4.57).
Increasing the light intensity increased the overall log reduction (3.27 vs 4.22).
Decreasing the volume of reaction mixture increased the log reduction. This study has
identified the best testing protocol for evaluating TiO2 NP bactericidal efficacy as single
wash of bacterial cells with a reaction mixture volume of 20 mL and UVA light intensity
of 2 mW/ cm2. In addition, it was found that photocatalytic oxidation of organic dyes can
be used as a quick and easier way to screen bactericidal TiO2 NPs prior to actual
microbiological tests.
Keywords: TiO2; Nanoparticles; Photocatalyst; Bactericidal activity; E.coli O157:H7.
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1. Introduction
Advanced oxidation processes based on heterogeneous photocatalysis using
photocatalytic nanoparticles (NPs) is gaining popularity in food safety applications.
Heterogeneous photocatalysis utilizes light along with a semiconductor NPs to produce
reactive oxygen species (ROS) which can inactivate bacteria and degrade a wide range of
chemical contaminants (Mills & Le Hunte, 1997). Of the available semiconductor NPs
which can be used as photocatalysts, TiO2 is generally considered to be the best
semiconductor photocatalyst available at present (Mills & Lee, 2002) due to its strong
oxidizing power at ambient temperature and pressure, stable, non-toxic, cheap and readily
available. TiO2 has been approved by the Food and Drug Administration (FDA) for use in
human food, drugs, cosmetics, and food contact materials (Chorianopoulos et al., 2011).
TiO2 photocatalysts generate strong oxidizing power when illuminated with UV light of
wavelength less than 385 nm. TiO2-mediated photooxidation shows promise for the
elimination of microorganisms in areas where the use of chemical cleaning agents or
biocides is ineffective or is restricted by regulations such as pharmaceutical and food
industries (Skorb et al., 2008). In addition, TiO2 becomes superhydrophilic upon
irradiation with UV light and this functionality is reversible and depends on the light
exposure (Chen & Mao, 2007). These properties of TiO2 help to reduce the usage of
cleaning agents and to create shorter cleaning cycles in the food industry.
Since the photochemical sterilization of E. coli using Pt-TiO2 was reported by
Matsunga et al. (1985), TiO2 photocatalysts have extensively studied to disinfect a broad
spectrum of microorganisms including viruses, bacteria, fungi, algae, and cancer cells
(Kim et al., 2003). The bactericidal properties of TiO2 are attributed to the high redox
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potential of the surface species also known as ROS, such as hydroxyl radical (.OH),
superoxide radical (O2.-), hydrogen peroxide (H2O2) formed by photo-excitation. The type
and the source of TiO2 plays an important role during bacterial inactivation because the
rate of formation of ROS is a function of the particle size, crystalline phase, the
isoelectric point, and specific surface area of the nanostructure (Hitkova et al., 2012). On
the other hand, the biological parameters of the microorganisms such as microbial
species, growth phase, and initial cell density etc., are also important and photocatalytic
disinfection process may vary depending on the light intensity, the wavelengths and
experimental conditions (Hitkova et al., 2012). Several commercial photocatalytic TiO2
products are available on the market and notably, Degussa P25 TiO2, which is considered
a standard is often used for comparison in scientific experimentation for determining
photocatalytic activity (Mills & Le Hunte, 1997). Hoffmann et al (1995) have suggested
that the anatase/rutile structure of P25 promotes charge-pair separation and inhibits
recombination. The different electron-hole pair recombination lifetimes and interfacial
electron-transfer rate constants may be due to the different preparation methods of the
samples that result in different crystal defect structures and surface morphologies (Chen
& Mao, 2007).
Several studies claimed high bactericidal activity of synthesized and commercial
TiO2 NPs that are equivalent to Degussa P-25 (Kim et al., 2003; Hitkova et al., 2012).
However, none of these studies have considered important factors like bacterial cell
harvesting conditions and reaction mixture volume that may negatively influence the
results of photocatalytic disinfection. Hence developing a systematic testing protocol to
evaluate bactericidal efficacy of NPs helps proper selection of TiO2 NPs for food safety
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related applications. The overall objective of this study was to develop a systematic
testing protocol to determine the bactericidal efficacy of different TiO2 nanoparticles in
suspension. Specific objectives include: i) to determine the effect of type and source of
TiO2 NPs on bactericidal activity, ii) to determine the effect of cell harvesting conditions,
light intensity and volume of suspension on bactericidal activity.
2. Materials and Methods
2.1. Nanoparticles
A total three different types of commercial TiO2 NPs (referred as T1, T2 and T3) of
known characteristics (Table 3.1) were used in this study. Samples T1 and T2 were of
anatase crystal phase with 10-25 nm size and > 99 % purity. While sample T3 is mixture
of anatase/rutile phase (~80:20 wt %) with ~ 21 nm size and > 99.5 % purity. The details
of the nanoparticle characteristics as provided by the individual manufacturer are listed in
Table 3.1. Aqueous suspensions of TiO2 NPs at 1 mg/mL concentration were prepared by
sonication in water-bath (Model # FS30, Fisher Scientific, Waltham, MA, USA) for
about 1 h at 23°C using sterile deionized water. The suspensions were prepared fresh
every time prior to each photocatalytic disinfection experiment.
2.2. Bacterial strains and inoculum preparation
Five strains of E. coli O157: H7 isolated from different sources: E009 (beef),
EO932 (cattle), O157-1 (beef), O157-4 (human), and O157-5 (human) were used in this
study. All bacterial strains were stored at -70 °C in tryptic soy broth (TSB) (Difco,
Becton Dickinson, Sparks, MD, USA) containing 20 % glycerol. Prior to the experiment,
cultures were activated at least twice by growing them overnight in 10 mL of TSB at 37
°C. Later each bacterial stain was cultured separately in 10 mL of TSB and kept on a
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shaking incubator at 230 rpm and 37°C for 16 h. Following the incubation, cells were
harvested by sedimentation either once (4000 x g for 12 min) or resuspended and
sedimented three times (3200 x g for 10 min) in sterile phosphate-buffered saline (PBS,
pH 7.2) in order to determine the effect of cell harvesting conditions on the photocatalytic
disinfection efficacy. The harvested cells were re-suspended in 10 mL PBS and an equal
volume of each strain suspension was combined to obtain 10 ml of a five strain cocktail
containing approximately 108 CFU/mL. Cell concentration was adjusted by measuring
the absorbance of bacterial suspension at 600 nm using a UV/Vis spectrophotometer
(Beckman DU520, Beckman Coulter Inc., Brea, CA, USA) and confirmed by plating 100
µL portions of the appropriate serial dilution on tryptic soy agar (TSA) (Difco
Laboratories) plates incubated at 37 °C for 24 h.
2.3. Photocatalytic experiments
The photocatalytic disinfection experiments were carried out in a sterile glass petri-
dish (90 x18 mm2; diameter x depth) mounted on a magnetic stirrer (Model# H1190M,
Hanna Instruments, Smithfield, RI, USA) which were together placed in a photocatalytic
disinfection chamber (Fig. 3.1). Aqueous suspensions of TiO2 NPs of 9, 18 and 27 mL
volumes were added into the petri-dish along with 1, 2 and 3 mL of bacterial cultures,
respectively. In this way, the effect of volume of suspension (10, 20 and 30 mL) on the
photocatalytic bactericidal activity was investigated for each commercial TiO2 sample
individually. The initial concentration of the bacterial culture in the suspension was fixed
at approximately 107 CFU/mL. The petri-dish with bacteria-NP suspension was
illuminated with a UVA light system fitted with four 40 W lamps (American DJ®, Model
UV Panel HPTM
, LL-UV P40, Los Angeles, CA , USA) from the top under continuous
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stirring at medium speed with a magnetic stirbar (3.8 cm length x 1.25 cm diameter) (Fig.
3.1). The intensity of the light was measured using a UV radiometer (Peak sensitivity 365
nm, UVP®, Upland, CA, USA). The light intensity reaching the surface of the sample
was adjusted to either 1 or 2 mW/cm2 (±0.15) by changing the distance between the light
source and the sample. A positive (photocatalyst in dark) and a negative (without
photocatalyst under UVA light) control samples were also included. All the experiments
were conducted at room temperature using indoor air as oxidant. A sample of 1 mL was
withdrawn from the treatment solution at every 30 min for 3 h and added into 9 ml sterile
PBS. Appropriate serial dilutions of the samples were prepared and the surviving bacteria
from the control and treatments were enumerated by spiral plating 50 µl of each dilution
on TSA. The plates were incubated at 37 °C for 24 h, and colonies were counted and
recorded as log CFU per mL. It was noticed that under tested conditions, positive and
negative controls had negligible effect on log reduction.
Further, the photocatalytic activity of TiO2 samples was evaluated by degradation of
methylene blue (MB) solution. The photodecay rate was measured by using 20 mL of
TiO2 aqueous solution (1 mg/mL) saturated with MB dye (20 mg/L) in a petridish
illuminated with UVA light at 2 mW/cm2 under continuous stirring as described earlier.
A 3 mL sample was collected at every 60 min for 3 h and the TiO2 NPs were separated
by centrifuging the suspension at 4000 rpm for 15 min at 4°C. The photodecay rate of
MB was determined by measuring the absorbance of supernatant at 664 nm using UV-
Vis spectrophotometer. Residual concentrations of MB (mg/L) due to TiO2 photocatalytic
activity was calculated by using standard MB curve.
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2.4. Data analysis
All the experiments were replicated three times. Data were subjected to an analysis of
variance with a completely randomized factorial design. Statistical analysis was
performed using SAS (2008) General Linear Model procedure performed with SAS
Software Release 9.3 (SAS Institute). T-tests were used for pairwise comparisons. Least
significant difference of means tests were done for multiple comparisons, and all tests
were performed with a level of significance 0.05.
3. Results and discussion
3.1. Effect of type and source of TiO2
The reduction trend of TiO2 (T1, T2 and T3) samples using single wash of
bacterial cells, 20 mL bacteria-NP suspension and 2 mW/cm2 UVA light intensity over a
3 h photocatalytic disinfection treatment is shown in Fig 3.2. Under tested conditions,
Sample T3 exhibited the highest log reduction (5.78 CFU/mL) followed by T2 (2.59
CFU/mL) and T1 (1.81 CFU/mL), respectively. This indicates that the type and source of
TiO2 has played important role on the log reduction. In particular, the difference in the
log reduction among the tested samples might be attributed to the differences in the
surface characteristics of the individual TiO2 NPs (Table 3.1). Sample T1 and T2 used in
this study have wide particle size distributions of 10-25 nm and sample T3 has an average
particle size of 21 nm. Size of the particle is an important factor which influences the
quantum yield of ROS responsible for the photocatalytic disinfection. Changes in particle
size influence photoactivity through changes in surface area, light scattering and light
absorptivity. Gerischer (1995) demonstrated that the quantum yield increases with
decreasing illumination intensity and size of the particle during photocatalysis. Bui et al.
93
(2008) pointed out that the differences in particle size, surface area and charge among
different varieties of TiO2 in powder affect the photocatalytic efficiency. In the current
study, though samples T1 and T2 have similar size distribution (10-25 nm), T2 showed
more bactericidal activity compared to sample T1 under similar test conditions. This
difference could be attributed to the high particle specific surface area (SSA) of T2 (200-
240 m2/g) compared to T1 (50-150 m
2/g). Increasing the particle surface area provides
high relative OH- ion coverages for hydroxyl radical (OH
·) formation which is an
important ROS responsible for photocatalytic disinfection. In contrast, sample T3 with
mixed phase of anatase (~80%) and rutile (~20%), an average particle size of ~21 nm and
relatively low SSA (35-65 m2/g) showed higher bactericidal activity than T1 and T2.
As reported in several studies, high bactericidal activity of mixed phase of
anatase-rutile TiO2 powder, particularly Degussa P-25 (sample T3) could be attributed to
the generation of high amounts of ROS such as OH·, O2- and H2O2. This could be due to
effective charge separation and by avoiding electron-hole pair recombination during
photocatalytic disinfection process. Gumy et al (2006) demonstrated that neither a high
surface area nor a high aggregate size can be the sole properties of a TiO2 photocatalyst
leading to optimal E.coli inactivation. Another study reported that Degussa P-25 obtained
by flame pyrolysis (Degussa, 1997) is an effective photocatalyst for bacterial
inactivation. Bickley et al (1991) reported that the dynamic process of Degussa P-25
preparation would create a complex variety of multiphased particles characterized by a
juxtaposition of anatase with rutile phases. This condition results in increased charge
separation and slows down electron (e-) - hole (h
+) pair recombination rate which in-turn
results in high photocatalytic activity. Nguyen et al (2005) reported for the same mass of
94
NPs, dispersed particle sizes of a commercial TiO2 PC500 are approximately three times
larger than TiO2 P-25. As a result P-25 provides a larger surface area per unit weight for
contact with bacteria than PC 500 and exhibits improved efficiency. This indicates that
the preparation method of the TiO2, NP surface structure, size, and crystallographic
structure all played important roles during the interfacial charge transfer between TiO2
and E.coli leading to disinfection (Gumy et al, 2006). The results of current study further
support the importance of type of TiO2 on bactericidal activity. Degussa P-25 was found
to be most effective among tested commercial TiO2 NPs.
However, several studies that reported high bactericidal activity of other
synthesized or commercial TiO2 NPs did not accounted for the effect of other influencing
factors such as bacterial culture harvesting method and volume of suspension used during
photocatalytic disinfection. These factors are important and need to be considered when
selecting TiO2 NPs for food safety applications.
3.2. Effect of cell harvesting conditions
The reduction trend of samples T1, T2 and T3 with respect to number of cell
washes at 2 mW/cm2 UVA intensity and 20 mL reaction mixture volume was shown in
Fig 3.2. Using single wash of bacterial cells, sample T1 and T2 showed reductions of
only 0.14, 0.62 log CFU/mL after 120 min and 1.81, 2.51 log CFU/mL after 180 min
treatment, respectively. Both samples required at least 90 min for initiating
photocatalytic disinfection. Whereas, sample T3 being more reactive right after 30 min
treatment showed a reductions of 3.12 and 5.78 log CFU/mL after 120 and 180 min,
respectively. It should be noted that depending on the reactivity of NP there will be a lag
in bacterial cell killing. This is in part attributed to the time required for the ROS to react
95
with bacterial cell membrane and facilitate damage and destruction of intracellular
components and eventual death of cell. It is clearly visible that sample T3 is the most
reactive among the tested TiO2 samples.
However, increasing the number of cell washes from 1 to 3 showed significant
change in the reduction efficacy of all three TiO2 samples. Sample T1 and T2 started
killing bacterial cells from around 30 min (a decrease of initial reactivity time at 90 min
for 1 wash) and showed a reduction of 3.28 and 4.48 log CFU/mL in 3h respectively
which is about 81 and 78% respective increase from the reduction at 1 wash treatment.
Sample T3 is least benefited by increasing number of cell washes as it showed no
difference in the reduction potential at the end of 3 h treatment. However, sample T3
showed better reduction between the treatment times 90 (98% increase) and 120 (44%
increase) min when compared with 1 wash treatment. These results indicate possible
damage to the bacterial cell membrane during additional centrifugation/sedimentation
prior to the photocatalytic disinfection treatment might have enhanced reduction efficacy
of TiO2 NPs.
Peterson et al. (2012) stated that centrifugation in essence involves compacting
bacteria into a pellet, causing collisions against each other that result in shear forces on
the bacterial cell surface, which may lead to cell surface damage with a potential effect
on the outcome of surface-sensitive experiments. Gilbert et al. (1991) reported a decrease
of 25 and 40% in the viability of exponential-phase Pseudomonas aeruginosa following
centrifugation at 5,000 x g and 10,000 x g respectively. Similar experiments with
stationary and exponential phase E. coli cells greatly altered biocide sensitivity (Gilbert et
al., 1990). Pembery et al. (1999) reported loss of viability and modification of
96
physicochemical cell surface properties of E. coli or S. epidermis by high-speed
centrifugation (15,000 x g) when compared to harvesting at (5000 x g). Similarly,
subjecting the bacterial cells to multiple washing steps in current study might have
damaged the outer cellular membrane and makes it more susceptible to ROS attack
during photocatalytic disinfection treatment. Especially, increasing the number of cell
washes resulted higher bactericidal efficacies by less effective commercial samples T1
and T2 (Fig 3.2). This situation may leads to false prediction of bactericidal efficacy of
TiO2 NPs while selecting NPs for food safety applications such as coating on food
contact and non-food contact surfaces. The results of this study suggest that bacterial
harvesting conditions are at-most important to accurately determine bactericidal activity
of photocatalytic NPs. It is recommended to use less severe harvesting conditions
depending on the type of test organism.
3.3 Effect of light intensity
Effect of UVA light (365 nm peak wave length) intensity on the log reduction of
E.coli O157:H7 due to TiO2 samples (T1, T2 and T3) is shown in Table 3.2. Increasing
the light intensity from 1 mW/cm2 to 2 mW/cm
2 increased the bacterial cell reduction
from 3.27 to 4.22 log CFU/mL. Although the trend of bactericidal efficacy was
unchanged (i.e. T3>T2>T1), the intensity of UVA light was shown to have a significant
effect on efficacy of individual TiO2 samples. Increasing the UVA intensity from 1 to 2
mW/cm2 significantly increased the log reduction of bacterial cells by T1 (2.04 to 3.04
CFU/mL), T2 (2.99 to 3.94 CFU/mL) and T3 (4.77 to 5.67 CFU/mL). Benabbou et al.
(2007) reported a decrease in light intensity from 3.85 to 0.48 mW/cm2
increased the time
necessary to totally inactivate E. coli (3 log) from 90 to 180 min. Increasing the light
97
intensity increases the amount of photon generation which results in more electron-hole
pair formation, eventually leading to the formation of more OH radicals (Marugan et al.,
2010). On the other hand, Cho et al. (2004) reported the existence of linear negative
correlation between inactivation of E.coli and OH concentration. This linear dependence
of reaction rate with the photon flux is only found at low intensities of irradiation,
because at high intensities the concentration of charge carriers is so high that
recombination is more favored, limiting the efficiency of the process (Hermann, 1999).
However, it is believed that the tested intensity range (1-2 mW/cm2) of the current study
operated in the linear region corresponding with optimal light utilization. This implies
that the saturation of TiO2 acting as the photosensitizer was not reached when increasing
the light intensity from 1 to 2 mW/cm2. Benabbou et al. (2007) noticed an induction
period in the first 10 min for lower light intensities, which suggests that self-defense and
auto-repair mechanisms for protecting the bacteria were more efficient at a low intensity.
However, self-defense mechanism unable to protect bacterial cells over a long treatment
time under the light. Similar results were also observed in the current study with an initial
lag period of ~30 min at the lower intensity (i.e. 1 mW/cm2) for photocatalytic
disinfection (data not shown). To further evaluate the photocatalytic disinfection efficacy
of TiO2 NPs, the effect of suspension volume was also investigated.
3.4. Effect of volume of suspension
Effect of volume of TiO2 NP-bacterial suspension (10, 20 and 30 mL) on
photocatalytic bactericidal activity for 3 h was shown in Table 3.2. Statistical analysis of
the data revealed that the overall log reduction of TiO2 is higher (4.32 CFU/mL) at lower
volume (10 mL) when compared to higher volumes (20 and 30 mL). No significant
98
difference in the reduction was observed between 20 and 30 mL volume of suspensions at
the end of 3 h treatment time. As expected, all the tested TiO2 samples (T1, T2 and T3)
exhibited high bactericidal activity at lower volume of suspension (10 mL). However,
volume does not showed any effect on overall reduction potential of least effective TiO2
sample T1 at the end of 3 h (avg ~ 2.5 log CFU/mL). Whereas, sample T2 showed
significantly higher reduction (4.30 log CFU/mL) at 10 mL and almost similar reductions
at 20 and 30 mL (~ 3 log CFU/mL). The most effective sample, T3 only showed
significant difference in the reduction between 10 mL (5.64 log CFU/mL) and 30 mL (4.7
log CFU/mL).
These results indicate that volume has significant effect on the bactericidal
activity of individual TiO2 NPs. We expected the improved efficacy of the least effective
TiO2 sample (T1) by optimal light utilization at lower volume of 10 mL. However, as per
the current study, no significant difference in the log reduction was observed by
decreasing the suspension volume from 30 mL to 10 mL for least effective sample T1.
Even though it is beyond the scope of our study to understand the agglomerate size of
NPs in suspension, its effect on ROS generation potential of individual TiO2 samples
should not be ruled out. If agglomerates were formed even providing more light flux by
decreasing the volume of suspension it will not improve photocatalytic disinfection
efficacy. To support this hypothesis, Gumy et al. (2006) reported that out of several
surface properties, aggregate sizes of several commercial NPs in suspension played an
important role during the interfacial charge transfer between TiO2 and E. coli leading to
bacterial abatement. Agglomerated condition reduces effective surface area of NPs
available for bacteria to come in-contact with while the suspension is stirred during
99
photocatalytic disinfection. Further we observed, a minimum of a 20 mL volume of
suspension is necessary for effective mixing of NP and bacteria while conducting
bactericidal efficacy tests in suspension.
3.5. Comparison of photocatalytic oxidation and photocatalytic disinfection rates of TiO2
The photocatalytic degradation rate of MB by three commercial TiO2 samples is
shown in Fig 3.3. Under tested conditions, sample T3 was most efficient (26% decay)
followed by sample T2 (10%) and T1(1%) in the degradation of MB solution by
photocatalytic oxidation. Similar photocatalytic disinfection trends in TiO2 samples (T1,
T2 and T3) were observed for E.coli inactivation (Fig 3.3). This shows that the
photocatalytic degradation potential of organic compounds such as MB can be used as a
prior screening test to indirectly predict bactericidal efficacy of TiO2 NPs since the
mechanism of both the processes depend on ROS generation rate. Chen et al. (2009)
reported apparent correlation between the photocatalytic processes of decomposing
formaldehyde and inactivating E.coli. They noticed a similar trend with respect to key
parameters such as light intensity, initial concentration, and type of nanomaterial on the
effect of photodegradation and disinfection. This study concluded analogously that this is
potential method to evaluate the antimicrobial effect based on organic compound
degradation. Similarly, Marugan et al. (2010) reported that similarities between
photocatalytic degradation of chemicals and microorganism inactivation which were
agreeable only when analyzing the effect of operational variables such as catalyst
concentration or light intensity. However, different microbiological aspects (osmotic
stress, repairing mechanism, regrowth, bacterial adhesion to TiO2 surface, etc) makes
disinfection kinetics significantly much more complex than the oxidation of chemical
100
compounds (Marugan et al., 2010). Hence certain similarities and differences exist
between photocatalytic oxidation and photocatalytic disinfection. However,
photocatalytic degradation of organic dyes such as MB can be used a quick and easier
test to screen bactericidal TiO2 NPs for food safety applications prior to actual
microbiological tests.
4. Conclusions
Photocatalytic disinfection efficacy of three different commercial TiO2 NPs to
inactivate E.coli O157: H7 has been systematically investigated. Type and source of TiO2
has showed significantly effect bacterial log reduction. Among the tested commercial
TiO2 samples, T3 (Degussa P-25) was found to be the most efficient photocatalyst
followed by T2 and T1. The same trend has been observed for photocatalytic degradation
of MB solution. Increasing the number of bacterial cell washes from 1 wash to 3 washes
prior to photocatalytic disinfection treatment increased the log reduction of even the least
effective TiO2 samples. It is preferred to use less severe cell harvesting conditions for
accurate determination of bactericidal efficacy of TiO2 NPs. As expected, increasing the
light intensity increased the bactericidal efficacy all TiO2 samples. Volume of suspension
showed variable effect on the efficacy of tested TiO2 NPs. As per the current study, using
20 mL of suspension with single wash of cells at less severe harvest conditions and 2
mW/cm2 UVA intensity was found to be best testing protocol for evaluating bactericidal
efficacy of TiO2 NPs.
Acknowledgements
Funding for this study was provided by the Agriculture and Food Research
Initiative grant no 2011-68003-30012 from the USDA National Institute of Food and
101
Agriculture, Food Safety: food Processing Technologies to Destroy Food-borne
Pathogens Program- (A4131).
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104
Table 3.1. Characteristics of commercial TiO2 NPs
a Commercial TiO2 samples T1: Sky Spring Nanomaterial’s; T2: US Research
Nanomaterials; T3: Degussa P-25 from Aldrich
TiO2
samplea
Crystal phase Purity
(%)
Size
(nm)
Specific Surface area
(m2/g)
True density
(g/cm3)
T1 Anatase 99.5 10-25 50-150 NA
T2 Anatase >99 10-25 200-240 3.9
T3 Anatase- Rutile ≥99.5 ~21 35-65 NA
105
Table 3.2. Effect of light intensity and volume on bactericidal activity of TiO2 NPs
Variable Log reductiona (CFU/mL) after 3 h treatment
Intensity (mW/cm2) Over all T1 T2 T3
1 3.27B
2.04B
2.99B
4.77B
2 4.22A
3.04A
3.94A
5.67A
Volume (mL)
10 4.32A 3.02
A 4.30
A 5.64
A
20 3.55B 2.32
A 3.09
B 5.32
AB
30 3.36B
2.28A
3.01B
4.70B
a Mean values with the same superscript in the same column within the same
variable combination are not significantly different (p >0.05)
106
Fig 3.1. Schematic of photocatalytic disinfection set-up; 1. Wooden chamber; 2. UVA
light bulbs (40 W each); 3. Magnetic stirrers; 4. Glass petridish with stirbar; 5. Height to
adjust light intensity.
2
4
3
1
5
107
Fig 3.2. Effect of TiO2 source and bacterial cell harvesting conditions on the log
reduction.
UVA: Without NP UVA alone; T1: Sky Spring Nanomaterial’s; T2: US Research
Nanomaterials; T3: Degussa P-25.
0
1
2
3
4
5
6
7
0 30 60 90 120 150 180
UVA(1 and 3 washes)
T1: 1 wash
T2: 1 wash
T3: 1 wash
T1: 3 wash
T2: 3 wash
T3: 3 wash
Log
red
uct
ion
(C
FU
/mL
)
Treatment time (min)
108
Fig 3.3. Comparison of photocatalytic degradation of methylene blue and photocatalytic
disinfection rate of E.coli O157:H7 among different TiO2 NPs.
T1: Sky Spring Nanomaterial’s; T2: US Research Nanomaterials; T3: Degussa P-25
0
1
2
3
4
5
60.7
0.75
0.8
0.85
0.9
0.95
1
0 30 60 90 120 150 180
Log r
edu
ctio
n,
CF
U/m
L
Met
hyle
ne
blu
e d
ecay, C
/C 0
Treatment time (min)
T1
T2
T3
T1
T2
T3
Photo disinfection
Photo oxidation
109
CHAPTER 4
EFFECT OF FOOD PROCESSING ORGANIC MATTER ON PHOTOCATALYTIC
BACTERICIDAL ACTIVITY OF TITANIUM DIOXIDE (TIO2)2
2Veerachandra K. Yemmireddy and Yen-con Hung. International Journal of Food
Microbiology 204 (2015) 75-80. Reprinted here with permission of the publisher.
110
Abstract
The purpose of this study was to determine the effect of food processing organic matter
on photocatalytic bactericidal activity of titanium dioxide (TiO2) nanoparticles (NPs).
Produce and meat processing wash solutions were prepared using romaine lettuce and
ground beef samples. Physico-chemical properties such as pH, turbidity, chemical
oxygen demand (COD), total phenolics (for produce) and protein (for meat) content of
the extracts were determined using standard procedures. The photocatalytic bactericidal
activity of TiO2 (1 mg/mL) in suspension with or without organic matter against
Escherichia coli O157:H7 (5-strain) was determined over a period of 3 h. Increasing the
concentration of organic matter (either produce or meat) from 0% to 100% resulted in
85% decrease in TiO2 microbicidal efficacy. Turbidity, total phenolics, and protein
contents in wash solutions had significant effect on the log reduction. Increasing the total
phenolics content in produce washes from 20 to 114 mg/L decreased the log reduction
from 2.7 to 0.38 CFU/mL, whereas increasing the protein content in meat washes from
0.12 to 1.61 mg/L decreased the log reduction from and 5.74 to 0.87 CFU/mL. Also, a
linear correlation was observed between COD and total phenolics as well as COD and
protein contents. While classical disinfection kinetic models failed to predict, an
empirical equation in the form of “Y=menX
” (where Y is log reduction, X is COD and m
and n are reaction rate constants) predicted the disinfection kinetics of TiO2 in the
presence of organic matter (R2=94.4). This study successfully identified an empirical
model with COD as a predictor variable to predict the bactericidal efficacy of TiO2 when
used in food processing environment.
Keywords: TiO2; Bactericidal activity; Organic matter; Kinetics; E.coli O157:H7
111
1. Introduction
More than two thirds of all fresh water abstraction worldwide goes toward food
production (Kirby et al., 2003). From the primary production of food to subsequent
processing requires copious amounts of water. One challenge for the food industry is to
minimize water consumption and waste water discharge rates (Olmez and Kretzschmar,
2009). Current trends toward sustainable production practices necessitate food industry
to reuse the water after proper treatment. However, it should be noted that water serves as
a source of cross-contamination as reusing processing water may result in the buildup of
microbial loads, including undesirable pathogens from the crop (Gil et al., 2009). Several
recent outbreaks related to foods can be traced back to contaminated process wash water
and irrigation water with pathogens. This shows inadequacy of existing physical and
chemical disinfection technologies.
Among several water disinfection technologies, chlorination is the most
extensively used for the last three decades (Pigeot-Remy et al., 2012). However, studies
show that in many cases chlorinated water is not fully effective in reducing pathogens
(Zhang et al., 2009) and has potential to generate harmful chlorinated disinfection by-
products like trihalomethanes, haloacetic acids, haloketones, and chloropicrin in presence
of organic matter (Gil et al., 2009; Lopez-Galvez et al., 2010). Moreover, pathogens such
as viruses, protozoa, or helminthes are generally more resistant to chlorine than bacteria
by varying degrees (Kirby et al., 2003). Other commonly used treatments such as
ozonation and filtration also have certain inherent limitations. In this context, advanced
oxidation processes (AOPs) involving photocatalytic nanoparticles (NPs) are gaining
popularity as a viable alternative to existing disinfection technologies.
112
Among various photocatalysts, titanium dioxide (TiO2) has been extensively
studied in the last 25 years for its photocatalytic disinfection properties (Hitkova et al.,
2012). TiO2 photocatalysts generate strong reactive oxygen species (ROS) such as the
hydroxyl radical (·OH), superoxide radical (O2.-), and hydrogen peroxide (H2 O2) when
illuminated with UV light with a wavelength of less than 385 nm. The photogenerated
ROS has proven to exhibit excellent microbicidal activity and is responsible for
mineralization of organic compounds. TiO2 is non-toxic and has been approved by the
American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics,
and food contact materials (Chawengkijwanich and Hayata, 2008). Bactericidal and
fungicidal effects of TiO2 on Escherichia coli, Salmonella choleraesuis, Vibrio
parahaemolyticus, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus
aureus, Diaporthe actinidiae, and Pencillium expansum have been discussed by Foster et
al. (2011). Among several commercial and synthesized TiO2 NPs, Degussa P-25 is
considered as a standard for determining photocatalytic activity (Mills and Le Hunte,
1997).
Several studies in the past have explained the disinfection mechanism of TiO2
(Foster et al., 2011) and explored the effect of nanoparticle size, concentration, UV light
intensity, pH, bacterial cell concentration, inorganic salts, and model organic matter on
the disinfection properties of TiO2 (Rincon and Pulgarian, 2004). However, the effect of
food processing organic matter on the bactericidal activity of TiO2 NPs is not well
reported. In general, the majority of the research studies concerning the evaluation of
sanitizing agents on the reduction of pathogenic microorganisms during washing do not
take into account the presence of organic matter (Stopforth et al., 2008). When potable
113
water is used to evaluate different sanitizing agents, it might lead to unrealistic results
with no practical application (Gil et al., 2009). Meat and produce wash operations in food
processing industries release abundant phenolic, protein, and lipid rich organic matter
along with several viable or nonviable pathogenic and spoilage microorganisms. Any
study exploring the optimum conditions for inactivation of pathogens and the effect of
organic matter on photocatalytic disinfection properties of UV activated TiO2 would help
to apply these novel technologies in still unexplored sectors like food processing waste
water treatment. Also, identifying the disinfection mechanism in suspension consisting
organic matter would help to develop effective strategies while coating these NPs on
abiotic surfaces and packaging materials.
Hence, the overall objective of this study was to determine the effect of organic matter on
bactericidal activity of TiO2 NPs. Specific objectives include the following:
i) To determine the bactericidal efficacy of TiO2 in wash water rich in phenolic and
protein contents
ii) To identify the factors those are most useful to predict the disinfection potential
of TiO2 NPs in real food processing environment
2. Materials and Methods
2.1.Preparation of wash water containing organic matter
Wash waters rich in organic matter representing produce and meat processing
operations were used in this study. Romaine lettuce was purchased from a local
supermarket (Griffin, GA, USA) and stored at 4°C until use. Any wilted and damaged
outer leaves of the lettuce were removed and discarded while internal leaves were cut into
about 2.5 cm2 pieces using clean and sterile scissors. Subsequently, 50 g of lettuce were
114
placed into stomacher bags (Whirl Pak®) containing 200 mL sterile deionized water, and
the mixture was homogenized for 2 min in a stomacher (Seward Stomacher®, 80
biomaster, Worthing, UK). Ground beef samples were prepared by separating lean and
visible fat portions from primal cuts of beef chuck (ExcelTM
, Cargill Meat Solutions
Corporation, Wichita, KS, USA). The separated lean meat portions were ground in a
meat grinder (LEMTM
, Size #8, West Chester, OH, USA) to obtain a near 100% lean
ground beef samples. Later, A 10 g sample of ground beef at different lean to fat weight
ratios (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) were weighed into a stomacher bag
containing 200 mL sterile deionized water and homogenized as described earlier. The
resultant extracts were filtered through a sterile Whatman® filter paper (No. 2, 185 mm
diameter, 8 µm pore size) and further diluted by a 1:2, 1:3, and 1:4 factor of lettuce or
beef extract to deionized water in order to provide different levels of the organic load.
These solution were referred as produce (lettuce extracts), and meat (beef extracts) wash
solutions and kept at 4 °C in darkness prior to use.
2.2. Analysis of wash water properties
The physico-chemical properties such as pH, turbidity, COD, total phenolics (for
produce) and protein (for meat) contents of the wash solutions were determined. The pH
of the samples was determined using a pH meter (Model # AR50, Fischer Scientific,
Pittsburgh, PA, USA). The turbidity was measured using a turbidity meter (Model #
19952, HF Scientific, Fort Myers, FL, USA) and expressed as nephelometric turbidity
units (NTU). The COD was determined by following reactor digestion method (Jirka and
Carter, 1975). Briefly, 1 mL of an appropriate dilution of the sample was added to the
COD reagent vial (P/N# TT20711, Orbeco, Sarasota, FL, USA) and the contents were
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mixed thoroughly. The samples were digested for 2 h on a heating block preheated to 150
°C. The digested samples in the vials were cooled down to room temperature, and the
COD values were read on a colorimeter (Model # DR/890, HACH®, Loveland, CO,
USA) and expressed as mg/L.
Total phenolic content of produce wash solution was determined using the Folin-
Ciocalteu assay as outlined by Singleton and Rossi (1965). One milliliter of sample was
added to 70 mL deionized water in a 125 mL screw cap bottle then 5 mL Folin-
Ciocalteu’s phenol reagent (Sigma Aldrich Co., St Louis, MO, USA) was added to the
solution. After thorough mixing, 15 mL of 20% (w/v) sodium carbonate solution was
added followed by enough water to bring the total volume to 100 mL. The mixtures were
sealed and incubated for at least 2 h at room temperature. The samples were then read at
750 nm in a 1 cm quartz cuvette using a DU 520 UV/Vis spectrophotometer (Beckman
Coulter Inc., Brea, CA, USA). The total phenolic content of a test sample was calculated
using catechol as a standard and reported as mg/L.
Total protein content of meat wash solution was determined using the Bradford assay
(Bradford, 1976). Briefly, 0.1 mL of sample was mixed with 5 mL Bradford’s reagent
(Sigma- Aldrich Co., St Louis, MO, USA). The samples were then read at 595 nm in a 1
cm quartz cuvette using a DU 520 UV/Vis spectrophotometer mentioned earlier. The
total protein content of a test sample was calculated using bovine serum albumen as a
standard and reported as mg/L.
2.3. Bacterial strains and inoculum preparation
Five strains of E. coli O157: H7 isolated from different sources: E009 (beef),
EO932 (cattle), O157-1 (beef), O157-4 (human), and O157-5 (human) were used in this
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study. All bacterial strains were stored at -70 °C in tryptic soy broth (TSB) (Difco,
Becton Dickinson, Sparks, MD, USA) containing 20% glycerol. Prior to the experiment,
cultures were activated at least twice by growing them overnight in 10 ml of TSB at 37
°C. Later, each bacterial stain was cultured separately in 10 ml of TSB and kept on a
shaking incubator at 230 rpm and 37°C for 16 h. Following the incubation, bacterial cells
were harvested by sedimentation at 4000 xg for 12 min and re-suspended in a sterile
phosphate-buffered saline (PBS, pH 7.2), and equal volumes of each strain suspension
were combined to obtain 10 mL of a five strain cocktail containing approximately 108
CFU/mL. Cell concentration was adjusted by measuring the absorbance of bacterial
suspension at 600 nm using a UV/Vis spectrophotometer and confirmed by plating 100
µL portions of the appropriate serial dilution on tryptic soy agar (TSA) (Difco
Laboratories) plates incubated at 37 °C for 24 h.
2.4. Photocatalytic disinfection experiments
TiO2 NPs (Aeroxide® P25, Sigma-Aldrich, St. Louis, MO, USA) with a surface
area of 50 m2 g
-1 and a particle size of ~21 nm (as per supplier specifications) were used
in this study. Suspensions of TiO2 (1 mg/mL) in produce and meat wash solutions were
prepared by sonication in water-bath (Model # FS30, Fisher Scientific, Waltham, MA,
USA) for about 1 h at 23°C. Photocatalytic disinfection experiments were carried out by
adding 2 mL bacterial culture in 18 mL NP suspension at 2 mW/cm2 UVA light intensity
by following method of Yemmireddy and Hung (2015). Briefly, the procedure involves,
20 mL bacteria-NP suspension, which was added into a sterile glass petri-dish (90x18
mm2; diameter x depth) mounted on a magnetic stirrer (Model# H1190M, Hanna
Instruments, Smithfield, RI, USA) and illuminated with a UVA light system fitted with
117
four 40 W lamps (American DJ®, Model UV Panel HP
TM, LL-UV P40, Los Angeles, CA,
USA) from the top under continuous stirring. The intensity of the light was measured
using UV radiometer (Peak sensitivity 365 nm, UVP®, Upland, CA, USA). A control
sample of bacterial culture suspended in wash water without photocatalyst under UVA
light was also included. All the experiments were conducted at room temperature using
indoor air as oxidant. A 1 mL sample was withdrawn from the treatment solution at every
1h for 3 h and added into 9 mL sterile PBS. Appropriate serial dilutions of the samples
were prepared, and the surviving bacteria from the control and treatments were
enumerated on Sorbitol Macconkey Agar (SMAC). The plates were incubated at 37 °C
for 24 h, and the colonies were counted and recorded as log CFU per mL. All the
experiments with produce wash were replicated five times and meat wash were
duplicated.
2.5. Kinetic models
The kinetics of photocatalytic bacterial inactivation is usually described using
empirical equations. The following five well-known disinfection kinetic models were
considered in order to find a best-fit model for the experimental results involving
photocatalytic bactericidal activity of TiO2 in the presence of organic matter:
The Chick-Watson model (Chick, 1908; Watson, 1908) with a constant
concentration of photocatalyst:
log (N/N0) = -k t (1)
where N/N0 is the reduction in bacterial concentration, k is the kinetic constant of
inactivation, and t is the treatment time.
118
The delayed Chick-Watson model (Cho et al., 2004) to accommodate if there
exists any initial lag time (t0) in the disinfection is computed as follows:
log (C/C0) = 0 for t ≤ t0
- k (t-t0) for t > t0 (2)
The modified Chick-Watson model (Cho et al., 2003) to accommodate either the
existence of a shoulder at the beginning of the reaction or a tail at the end of the reaction:
log (C/C0) = k1[1-exp(-k2t)] (3)
The Homs model (Hom, 1972) when the inactivation rate deviates log-linear
behavior is calculated as follows:
log (C/C0) = - k th (4)
Where h is the second parameter. If h=1, Homs model becomes a Chick-Watson linear
equation, h>1 for existence of a shoulder, h<1 for existence of a tail.
The modified Homs model (Cho et al., 2003) to accommodate shoulder, log-
linear, and tail regions is calculated as follows:
log (C/C0) = k1[1-exp(-k2t)]k3
(5)
To determine which model best described the data, the estimated coefficient of
determination (R2) and the F-value were calculated using the following equations:
Residual sum of squares
R2
= 1 - (6)
Uncorrected total sum of squares
Mean regression sum of squares
F
= (7)
Mean squared error
119
3. Results and discussion
3.1. Kinetics of TiO2 disinfection and the effect of organic matter
Fig. 4.1 shows the results of the photocatalytic disinfection of E. coli O157:H7
using TiO2 aqueous suspensions with different levels of organic matter from meat and
produce extract solutions. TiO2 in suspension without organic matter has showed a
reduction of around 5.78 log CFU/mL after 3 h treatment. While, TiO2 suspended in meat
and produce organic matter extracts at 25% level of incorporation in the reaction mixture
showed a reduction of only 3.7 and 2 log CFU/mL, respectively. Further increasing the
organic matter concentration to 100% in the reaction mixture significantly reduced the
disinfection potential of TiO2 to below 1 log CFU/mL. This shows that increasing the
organic matter content in the reaction mixture has detrimental effect on the TiO2
bactericidal activity. This can be explained based on the premise that the process of
decomposing organic matters and photo-killing of microbes is perceived to follow the
similar mechanisms of the attack by ROS (Chen et al., 2009). However, the organic
matter present in the reaction mixture competes with bacteria for hydroxyl radical (OH.),
which is a major ROS responsible for the killing of bacteria and also hinders the
interaction between the bacteria and the TiO2 catalyst (Grieken et al., 2010). The same
phenomena might be the reason for decreased bactericidal activity of TiO2 in the current
study. However, the effect of specific components of organic matter in the meat and
produce extract solutions on the photocatalytic disinfection efficacy of TiO2 need to be
further investigated.
The photocatalytic disinfection kinetics of TiO2 with or without organic matter
has followed a non-linear trend with a shoulder (Fig. 4.1). The experimental data were
120
fitted with most commonly used empirical models that are described earlier. When the
reaction mixture is free from any organic matter, almost all the tested empirical models
were able to fit the experimental data well with an R2
value greater than 0.94 (Table 4.1).
When considering both R2 and F-statistic values to predict the goodness of fit, only the
modified Chick -Watson and the modified Homs model were able to give the best fit with
an R2
value of 0.98 and F-value of 274.81. However, due to lack of a tail region at the end
of photocatalytic disinfection treatment, modified Homs model is insignificant to fit the
data obtained from the current study. Hence, the modified Chick-Watson model to
accommodate initial lag or shoulder effect was found to be the most appropriate model to
predict the disinfection kinetics of TiO2. With the incorporation of organic matter like
produce or meat extract in the reaction mixture, none of the tested empirical models were
able to fit the TiO2 disinfection data well. Only, the modified Chick-Watson model was
able to predict the disinfection trend of TiO2 for up to 25% level of organic matter from
both meat (R2= 0.993) and produce (R
2 = 0.933) extracts (Table 4.1). However, the
modified Chick-Watson model failed to predict the TiO2 disinfection kinetics when
organic matter concentration was more than 25%. This further supports the hypothesis
that effect of individual components of organic matter need to be accounted to better
predict the disinfection kinetics of TiO2 in the presence of organic matter.
3.2. Effect of pH
The effect of pH of produce and meat extract solutions on the disinfection
potential of TiO2 was shown in Table 4.2. Decreasing the organic load in wash solutions
from 100% to 25% increased the log reduction of TiO2 from 0.54 to 2.07 for produce and
0.87 to 3.7 for meat extract solutions, respectively. Although, the pH values of produce
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and meat extract solutions were significantly different from each other, they are not
different within the same type of extract solutions at different levels of organic load. In
both types of extracts, even at same level of pH, the log reductions are significantly
different from each other. For example, produce extract solution at concentration of
organic load 50% and 75% with pH 6.2 showed significantly different reductions of 1.4
and 0.74 log CFU/mL, respectively. This implies that under tested conditions, pH of the
solutions containing organic matter alone does not have an effect on the disinfection
potential of TiO2. Gumy et al. (2006) reported that the electrostatic attraction between the
E. coli and the Degussa P-25 TiO2 is not a controlling factor in the pH range of 4.5 to 6.0
since E. coli is negatively charged between pH 3 and 9 and TiO2 is positively charged up
to pH 7. In another study by Rincon and Pulgarin (2004), modification of pH of TiO2
suspension did not show any effect on the E.coli inactivation rate in the pH range of 4
and 9. Similarly, the pH range (5 to 6.27) of produce and meat extract solutions used in
the current study may not have an effect on the photocatalytic disnfection efficacy of
TiO2.
3.3. Effect of Turbidity
The effect of turbidity of produce and meat extract solutions on the disinfection
potential of TiO2 was shown in Fig. 4.2. Increasing the turbidity of produce extract from
36 to 148 NTU decreased the log reduction from 2.08 to 0.54 CFU/mL. Similarly,
increasing the turbidity of meat extract solutions from 17 to 50 NTU decreased the log
reduction from 3.7 to 0.38 CFU/mL. This shows that increasing the turbidity of wash
solutions decreased the bactericidal efficacy of TiO2. Turbidity caused by the presence of
components leaching from tissues of the cut produce surface and meat, is a measure of
122
the waters ability to scatter and absorb light, which depends on a number of factors such
as size, number, shape, and refractive index of the particles and the wave length of
incident light (WHO, 1996). The photogenerated ROS, such as hydroxyl radical are
highly active for both the oxidation of organic substances and the inactivation of bacteria
(Kim et al., 2003). Both the bacteria and the organic matter present in the suspension
compete for the ROS generated through photocatalytic disinfection process. This
condition reduces the disinfection potential of TiO2 to inactivate bacteria. In addition,
increasing the turbidity of the reaction mixture decreases the penetration power of UVA
light into the solution and limits the ability of TiO2 NPs to generate ROS. Selma et al.
(2008) studied the turbidity effect of various fresh-cut vegetable wash waters on the
disinfection potential of TiO2. Their study reported that differences in water turbidity
were associated with different bacterial inactivation rate. Onion wash water with highest
turbidity (5040 NTU) has least bacterial inactivation rate and carrot wash water with
lowest turbidity (0.6 NTU) has highest inactivation rate. However, lettuce (87.4 NTU),
escarole (95.7 NTU), chicory (42.4 NTU) and spinach (88.9 NTU) wash waters with
intermediate level of turbidity have showed lower bacterial inactivation. In our study,
upon gradual decrease in the lean to fat ratio of meat extract solution from 100:0 to 0:100
resulted in almost 18% to 40% decrease in the turbidity (results not shown). However, no
significant increase in the bactericidal activity of TiO2 was observed. This clearly shows
that turbidity itself is not a rate limiting factor and the presence of other components of
the organic matter such as fat content may affect the bactericidal efficacy of TiO2. The
presence of components such as protein and fat in the reaction mixture might have
blocked the surface active sites on TiO2 NPs to generate ROS and reduced the efficiency
123
of photocatalytic disinfection process. This implies that the efficacy of the photocatalytic
system will be highly depend on the physicochemical characteristics of the suspension
containing organic matter and increasing the turbidity of the suspension reduced the
photocatalytic inactivation rate of bacteria.
3.4. Effect of total phenolics and its correlation with COD
Fig. 4.3 presents the effect of total phenolics content in the produce extract on
photocatalytic bactericidal activity of TiO2. Total phenolics content in the suspension
showed significant effect on the bacterial inactivation. For example, increasing the total
phenolics content in the suspension from 20.4 to 113.6 mg/L decreased the log reduction
from 2.7 to 0.38 CFU/mL. The reduction trend can be best fitted with an exponential
equation in the form of Y = A eBX
(where Y is the log reduction in CFU/mL, X is the total
phenolics in mg/L, and A and B are constants) with an R2 value of 0.943. Also, a linear
correlation was observed between total phenolics and COD of the produce extract
solution (Fig 4.3). It followed a regression trend of Y=40.22X-220.77 (where Y = COD in
mg/L, X = Total phenolics in mg/L) with a correlation coefficient 0.928. One possible
reason for the decreased photocatalytic activity of TiO2 can be attributed to the increased
concentration of phenolic compounds in the suspension. Phenolic compounds such as
tocopherols, flavonoids, and phenolic acids are well known for their antioxidant activity.
In general, these compounds inhibit or delay the oxidation of other molecules by
inhibiting the initiation or propagation of oxidizing chain reactions. TiO2 photocatalysis,
which involves series of oxidation and reduction reactions, is highly dependent on the
generation of ROS. The phenolic compounds present in the produce extract might have
quenched the generated ROS by irradiated TiO2 NPs, which in turn reduced its efficacy to
124
inactivate bacteria. Rincon and Pulgarin (2004) reported a significant decrease in the
TiO2 photocatalytic inactivation of E. coli in the presence of organic compounds such as
dihydroxybenzenes, hydroquinone, catechol, and resorcinol. They reported that the
formation of an optical screen on TiO2 surface by organic and inorganic components for
light penetration as well as competition of organic compounds for OH radicals are some
reasons for decreased photocatalytic efficacy. Similar phenomena can be attributed to the
decreased bactericidal activity of TiO2 in the presence of phenolic-rich organic matter
used in the current study.
3.5.Effect of protein and its correlation with COD
The effect of protein content in meat extract on the log reduction was shown in
Fig. 4.4. As expected, increasing the protein content from 0.12 to 1.61 mg/L in the
reaction mixture decreased the log reduction from 5.74 to 0.84 in mg/L. The reduction
trend can be represented with an exponential equation in the form of Y = A eBX
(where Y
is the log reduction in CFU/mL, X is the protein content in mg/L, and A and B are
constants) with R2 value of 0.904. Like phenolics, a linear correlation between protein
and COD of the meat extract was noticed with an R2
value of 0.725 (Fig 4.4). Variable
proportions of lean to fat ratios (100:0 to 0:100), and the relative complexity of meat
extract might be one possible reason for the distorted trend and poor correlation of the
protein with log reduction and COD. In general, TiO2 NPs tend to agglomerate in
aqueous solutions in the absence of agitation. The presence of organic matter rich in
protein further enhances the formation of agglomerated NPs in suspension irrespective of
agitation. In addition, it is possible that the fat molecules present in the meat extract
forms an outer layer on the surface of TiO2 NPs, which results in blockage of surface
125
active sites for the photocatalytic reaction to takes place and subsequent generation of
ROS. Gumy et al. (2006) reported that out of several surface properties, the aggregate
size of several commercial NPs in suspension played an important role during the
interfacial charge transfer between TiO2 and E. coli leading to bacterial abatement.
Agglomerated condition reduces the effective surface area of NP available for bacteria to
come incontact with while stirring the suspension during photocatalytic disinfection. The
same might be the reason for the decreased bacterial inactivation rate of TiO2 in the
presence of organic matter rich in protein. However, further studies need to be conducted
to understand the effect of individual components on photocatalytic disinfection
mechanism of TiO2 in complex food systems such as meat extract.
3.6. Effect of COD
Although, total phenolics and protein contents are reasonably good in predicting
the bactericidal efficacy of TiO2 in the presence of organic matter, using a common factor
such as the COD might be practically more beneficial. As discussed before, COD of
produce and meat extract solutions had a linear correlation with phenolics (R2= 0.92) and
protein contents (R2= 0.72), respectively. Hence, COD can be used as a predictor variable
to determine the kinetics of TiO2 bacterial inactivation in the presence of organic matter.
Fig. 4.5 shows the correlation between the COD of the produce or meat extract solutions
and the log reduction of E.coli O157:H7. Increasing the COD values of both meat and
produce extracts decreased the log reduction. Experimental data from both meat and
produce extract solutions were best fitted with an empirical model in the form of Y= menX
(where Y is the log reduction, X is the COD of organic matter, and m and n are reaction
rate constants) (Table 4.3). A study conducted by Selma et al, (2008) on different types of
126
produce wash waters reported that onion wash water with highest COD was associated
with the least bacterial reduction after treatment with the photocatalytic system.
According to these results, it appears that the inactivation data can be better correlated
with the COD of organic matter in the suspension. TiO2 photocatalytic action was
attributed to the promotion of peroxidation of phospholipid components of the lipid
membrane, inducing cell membrane disorder, subsequent loss of essential functions such
as respiratory activity, and cell death (Ibanez et al, 2003). The generation of hydroxyl
radical induced by UV radiation rapidly overcomes the self-protection mechanisms of the
bacterial cell, and as a result microbial counts decrease exponentially. In the last period of
photo-treatment, the rate of microbial inactivation becomes slower because OH radicals
produced by the irradiated TiO2 act against the few active bacteria remaining in the UV-
irradiated water but also against the inactivated bacteria and the metabolites released
during the photocatalytic treatment (Rincon and Pulgarian, 2003). A similar mechanism
can be attributed to the decrease in photocatalytic bactericidal efficacy of TiO2 NPs in the
presence increasing levels of organic matter.
4. Conclusions
The results of this study showed that the presence of organic matter from both
produce and meat extract solutions has a significant effect on the bactericidal efficacy of
TiO2. Under tested conditions, the pH level of the produce and meat wash solutions had
no significant effect on the bactericidal activity of TiO2, whereas turbidity, COD, total
phenolics, and protein content had a significant effect on the bactericidal efficacy of
TiO2. A linear correlation was observed between COD and total phenolics as well as
COD and protein content. While classical disinfection models failed to predict, an
127
empirical equation with COD as predictor variable successfully fit the experimental data.
The empirical equation proposed in this study helped to predict the photocatalytic
disinfection efficacy of TiO2 in the presence of food processing organic matter.
Acknowledgments
Funding for this study was provided by the Agriculture and Food Research
Initiative grant no 2011-68003-30012 from the USDA National Institute of Food and
Agriculture, Food Safety: Food Processing Technologies to Destroy Food-borne
Pathogens Program-(A4131).
128
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Table 4.1. Comparison of kinetic models to predict the TiO2 disinfection efficacy with or
without organic matter
Type Model R2
F-statistic
TiO2 without organic matter Chick-watson 0.947 194.98
TiO2 without organic matter Delayed Chick-Watson 0.948 200.56
TiO2 without organic matter Hom's model 0.985 233.28
TiO2 without organic matter Modified Hom's model 0.982 274.81
TiO2 without organic matter Modified Chick-Watson 0.982 274.81
TiO2 with 25% produce extract Modified Chick-Watson 0.933 125.69
TiO2 with 25% meat extract Modified Chick-Watson 0.993 417.94
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Table 4.2. Effect of pH of wash solution containing organic matter on
the bactericidal activity of TiO2
Type Organic load
(vol%)
pHa
Reductiona at 3 h
(Log CFU/mL)
Produce 100 6.11A
0.54E
75 6.20A 0.74
E
50 6.20A
1.40CD
25 6.27A 2.07
B
Meat 100 5.30B 0.87
DE
75 5.04B 1.04
CDE
50 5.08B 1.60
CB
25 5.07B 3.70
A
a Mean values with the same superscript within the same column are
not significantly different (p >0.05).
133
Table 4.3. Comparison of fitted isotherm parameters of empirical model
Model equation Y= m e nX
; where Y= Log reduction (CFU/mL),
X = COD (mg/L), and m and n are reaction rate constants.
Type of organic matter m n R2 F -statistic
Produce 3.5181 -0.00065 0.972 315.37
Meat 7.7101 -0.00157 0.960 266.64
Combined 6.7116 -0.00134 0.944 353.92
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Fig 4.1. Effect of different levels of organic matter from produce and meat
extract solutions on the log reduction of E.coli O157:H7 by TiO2
photocatalysis.
0
1
2
3
4
5
6
7
0 30 60 90 120 150 180
Log r
educt
ion (
CF
U/m
L)
Time (min)
TiO2 without organic matter TiO2 with 25% produce wash
TiO2 with 100% produce wash TiO2 with 25% meat wash
TiO2 with 100% meat wash UVA only
135
Fig 4.2. Effect of turbidity of produce and meat extract solutions on the
log reduction of E.coli O157:H7 by TiO2 photocatalysis for 3h.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 15 30 45 60 75 90 105 120 135 150
Log r
educt
ion (
CF
U/m
L)
Turbidity (NTU)
Produce extract
Meat extract (100% lean)
136
Fig 4.3. Correlation between total phenolics and COD of produce extract as well
as total phenolics and log reduction of E.coli O157:H7 by TiO2 photocatalysis.
Y = 4.175 e-0.026X
R² = 0.943
y = 40.22x - 220.77
R² = 0.928
0
1000
2000
3000
4000
5000
0
0.5
1
1.5
2
2.5
3
20 30 40 50 60 70 80 90 100 110 120
CO
D (
mg/m
L)
Log r
educt
ion (
CF
U/m
L)
Total Phenolics (mg/L)
137
Fig 4.4. Correlation between protein content and COD of meat extract as well as
protein and log reduction of E.coli O157:H7 by TiO2 photocatalysis.
Y = 6.2625e-2.8211X
R² = 0.904
Y = 1088.5X + 403.1
R² = 0.725
0
500
1000
1500
2000
2500
0
1
2
3
4
5
6
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
CO
D (
mg/L
)
Log r
educt
ion (
CF
U/m
L)
Protein (mg/L)
138
Fig 4.5. Relationship between COD of produce and meat organic matter
extracts and the log reduction of E.coli O157:H7 by TiO2 photocatalysis.
0
1
2
3
4
5
6
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Log r
educt
ion (
CF
U/m
L)
COD (mg/L)
Produce extract
Meat extract
139
CHAPTER 5
METHOD DEVELOPMENT FOR CREATING TITANIUM DIOXIDE (TIO2)
NANOCOATINGS ON FOOD CONTACT SURFACES AND METHOD TO
EVALUATE THEIR DURABILITY AND PHOTOCATALYTIC BACTERICIDAL
PROPERTY3
3Veerachandra K. Yemmireddy, Glenn D. Farrell and Yen-con Hung. Submitted to
Journal of Food Science, 2/9/2015.
140
ABSTRACT: Titanium dioxide (TiO2) is a well-known photocatalyst for its excellent
bactericidal property under UVA light. The purpose of this study was to develop
physically stable TiO2 coatings on food contact surfaces using different binding agents
and develop methods to evaluate their durability and microbicidal property. Several types
of organic and inorganic binders such as polyvinyl alcohol, poly ethylene glycol,
polyurethane, polycrylic, sodium and potassium silicates, shellac resin and other
commercial binders were used at 1:1 to 1:16 nanoparticle to binder weight ratios to
develop a formulation for TiO2 coating on stainless steel surfaces. Among the tested
binders, polyurethane, polycrylic, and shellac resin were found to be physically more
stable when used in TiO2 coating at 1:4 to 1:16 weight ratio. The physical stability of
TiO2 coatings was determined using adhesion strength and scratch hardness tests by
following standard ASTM procedures. Further, wear resistance of the coatings was
evaluated based on a simulated cleaning procedure used in food processing environments.
TiO2 coating with polyurethane at a 1:8 nanoparticle to binder weight ratio showed the
highest scratch hardness (1.08 GPa) followed by coating with polycrylic (0.68 GPa) and
shellac (0.14 GPa) binders. Three different techniques, namely direct spreading, glass
cover-slip, and indented coupon were compared to determine the photocatalytic
bactericidal property of TiO2 coatings against E.coli 0157:H7 at 2 mW/cm2 UVA light
intensity. Under the tested conditions, the indented coupon technique was found to be the
most appropriate method to determine the bactericidal property of TiO2 coatings.
Keywords: TiO2 coating; Food contact surface; Antimicrobial test; Binding agent;
Physical stability, E.coli.
141
Practical Application: A simple approach to create physically stable and bactericidal
TiO2 nanocoatings was developed on food contact surfaces of stainless steel using
different binding agents. The developed TiO2 nanocoatings might help to minimize
microbial cross-contamination and ensure safe food processing environment.
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Introduction
Microbial-cross contamination is a major issue in the food processing
environment leading to several foodborne outbreaks and illnesses in the recent times.
Cross contamination through both food contact and non-food contact surfaces such as
knifes, cutting boards, working surfaces, equipment and the processing environment is
well reported. Also, increased popularity of fresh and minimally processed foods poses
additional risk of cross-contamination from food contact and non-food contact surfaces
(Lloret and others 2012).
Conventional sanitation and disinfection procedures that are widely followed in
the industry are not sufficient to address the emerging risks of microbial cross-
contamination involving resistant pathogens. In addition, many of the established
sanitizers and disinfectants lack residual effect for extended period of protection and
generate toxic disinfection by-products (Meylheuc and others 2006). Modification of
surfaces with antimicrobial agents to prevent the growth of harmful microorganisms has
received much attention for several industrial applications (Rai and others 2010). In this
context, nanotechnology based advanced oxidation processes involving photocatalytic
titanium dioxide (TiO2) nanoparticles (NPs) have shown great promise as an effective
non-targeted disinfectants for killing a wide range of microorganisms.
Over the last decade, there is an increased interest in the application of TiO2
photocatalytic disinfection technique for the purpose of food safety and quality
enhancement (Manreet and Hayata 2006; Chawengkijwanich and Hayata 2008;
Chorianopoulos and others 2011). TiO2 has been recognized as the most promising
photocatalyst due to its appropriate electronic band structure, photostability, chemical
143
inertness, low cost, ready availability, and capable of repeated use without substantial
loss of catalytic activity (Ibhadon and Fitzpatrick 2013). TiO2 has been approved by the
American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics,
and food contact materials (Chorianopoulos and others 2011). TiO2 photocatalysts
generate strong oxidizing power when illuminated with UV-A light of wavelength less
than 385 nm. The bactericidal properties of TiO2 were attributed to the high redox
potential of the reactive oxygen species (ROS) such as hydroxyl radical (.OH),
superoxide radical (O2.-), and hydrogen peroxide (H2O2) formed by the photo-excitation
(Foster and others 2011). Several techniques have been proposed to immobilize TiO2 NPs
on hard surfaces for the purpose of photocatalytic disinfection (Visai and others 2011).
Sol-gel synthesis of NPs and subsequent dip, spin, or spray coating is the most
widely reported procedure in the literature. Other coating methods such as
electrochemical deposition, electrophoretic coating, chemical vapor deposition,
sputtering, and plasma spraying were complicated and costly for practical application
(Kasanen and others 2011). Alternatively, a simple direct coating of the NPs using wet
chemical approaches were also reported. However, poor adhesion on the substrate and the
lack of physical stability of the developed coatings is the major issue when using direct
coating methods (Mills and Lee 2002; Keshmiri and others 2004; Han et al, 2012). Thus,
binding agents are usually necessary for direct coating in order to form strong adhesion
between the NP and the substrate. Either organic polymer or inorganic binding materials
have been used for photocatalyst immobilization (Du and others 2008; Lim and others
2009; Henderson and others 2011). However, no extensive stability studies have been
conducted in the past to evaluate the durability of the nanocoatings when they are
144
intended to use in food processing environment. In addition, several methods have been
proposed to determine the bactericidal activity of photocatalytic nanocoatings with each
having their own advantages and disadvantages.
For use in food safety applications, the antimicrobial coatings expected to have
characteristics such as long lasting efficacy, ease of fabrication, durability, and no
toxicity. With appropriate binding agents, physically stable and durable TiO2
nanocoatings with strong bactericidal property can be developed on food contact
surfaces. Hence, the main goal of this study was to identify appropriate binders to create
stable TiO2 nanocoating on stainless steel surface and identify methods to evaluate
physical stability and bactericidal property of TiO2 nanocoating. Specific objectives
include:
i) To identify the most promising binders to create TiO2 coatings on stainless steel.
ii) To develop a simple method for coating TiO2 NPs on stainless steel.
iii) To evaluate the physical stability and the durability of the TiO2 coatings.
iv) To identify a suitable testing method to determine the bactericidal property of
TiO2 coatings.
Materials and Methods
Selection of TiO2 NPs and Binders for coating
TiO2 (Aeroxide®
P25, Sigma-Aldrich, St. Louis, MO, USA) NPs with an
approximate particle size of 21 nm and specific surface area of 50 m2 g
-1 as per suppliers
specifications were used for developing nanocoatings in this study. Ten different types of
polymeric, silicate, and resin based organic and inorganic binding agents: (i) Polyvinyl
alcohol (PVA) (TCI America, Portland, OR, USA), (ii) Polyethylene glycol (PEG) (TCI
145
America, Portland, OR, USA), (iii) Ludox AS-40 (LAS-40) (Sigma-Aldrich, Co., St.
Louis, MO, USA), (iv) Two types of potassium silicates (PS1& PS6) (KASIL®
1 &
KASIL®
6, PQ Corporation, Valley Forge, PA, USA), (v) Two types of sodium silicates
(SSO & SSN) (O® & N
®, PQ Corporation, Valley Forge, PA, USA), (vi) Ceramabind 830
(C830) (Aremco Products, Inc., Valley Cottage, NY, USA), (vii) Ceramabind 643-1
(C643-1) (Aremco Products, Inc., Valley Cottage, NY, USA), (viii) Water based oil-
modified polyurethane (B) (Miniwax®, Miniwax company, Upper saddle river, NJ,
USA), (ix) Water based polycrylic (C) (Miniwax®, Miniwax company, Upper saddle
river, NJ, USA), (x) Shellac (A) (Zinsser Co., Inc. Somerset, NJ, USA) were tested to
evaluate their feasibility to incorporate in coating solution to achieve physically stable
TiO2 coatings.
Substrate selection and preparation
Stainless steel (Type 304, finish #2B, 25 mm2) coupons were used as model food
contact surface for coating. Prior to coating, each coupon was slightly roughened to
increase coating adhesion and achieve a high level of bond strength by using an electric
sander fitted with a P100 fine grit sand paper for 1 min on each side of the coupon. Later,
the surface roughened coupons were degreased first by washing in acetone followed by
ethanol and finally rinsed with deionized water. The cleaned stainless steel coupons were
dried in a hot air oven at 60°C for 30 min before used for coating.
Screening of binders for developing stable TiO2 coating
Several preliminary experiments were conducted in order to select binding agents
that are most suitable to develop physically stable TiO2 coatings on stainless steel. In the
first stage of experiments, TiO2 coatings were prepared by using two types of organic
146
binders PVA and PEG. Several suspensions of TiO2 NPs mixed with PVA or PEG
binders at 1:1 to 1:5 NP to binder weight ratios were prepared by using ethanol as
solvent. Stainless steel coupons were dip coated with the resultant suspensions using an
Instron (Model #5544, Instron Corporation, Canton, MA, USA) operated with a dipping
speed of 10 mm/s, residence time of 10 s and a withdrawal speed of 0.5 mm/s. In this
manner single or multiple coatings of TiO2 were applied on each coupon based on the
viscosity of coating suspension and uniformity of the coated film. The coated coupons
were dried in a hot air oven at 60 °C for 1 h. The dried coupons were visually inspected
for coating uniformity and washed under running water for about 5 to 10 min in order to
quickly assess the adherence behavior and physical stability of the coatings. Even though
heat treatment after coating helped to increase the adherence of the coating to stainless
steel surface, heat treatment also resulted in formation of clumps on the TiO2
nanocoating. The results of these experiments showed that the TiO2 nanocoatings with
PVA and PEG as binders are non-uniform in nature and unable to withstand washing
under running water (data not shown).
In the second stage of coating experiments, inorganic binders such as Ludox AS-
40 (LAS-40), potassium (PS1 and PS6) and sodium (SSO and SSN) silicates from a
commercial source as well as two other commercial binders of unknown composition
(C830 and C643-1) were used for TiO2 nanocoating. Twenty different paste formulations
(5 different binders at 4 different NP to binder ratios) were prepared for coating by
mixing TiO2 NPs with each binder at 1:1 to 1:4 NP to binder weight ratios. About 1 g
(±0.15) of the TiO2 paste was weighed and painted on each coupon using a Crayola paint
brush so as to form a layer of uniform TiO2 coating. TiO2 coatings with potassium and
147
sodium silicates (PS1, PS6, SSO & SSN) and C830 were air-dried at room temperature
for about 1 h; while the TiO2 coatings with LAS-40 and C643-1 were air-dried at room
temperature for 2 h and then cured at 93°C for 1 h in a hot-air oven as per the
manufacturer guidelines. Increasing the concentration of binder (i.e. less NPs to binder
ratio), increased the viscosity of TiO2 pastes and physical stability of the resultant
coatings. TiO2 coatings with potassium and sodium silicates at 1: 4 weight ratio was
found to be physically more stable upon scratching with pencil points of 2H hardness.
However, these coatings were not stable upon washing in running water followed by
sonication in water bath for 15 min. Based on these results it was found that the TiO2
coatings with tested inorganic binders helped to achieve uniform and physically stable
coatings (data not shown). However, TiO2 coatings with these binders are not suitable for
application in moist conditions encountered in food processing environment.
In the third stage of coating experiments, polymer based sealers polyurethane (B)
and polycrylic (C), as well as a natural resin, shellac (A), were tested for their feasibility
to incorporate in TiO2 coatings. The composition of different TiO2 coatings with binders
A, B, and C are presented in Table 5.1. Based on the nature of each binder, the viscosity
of coating solution increased with decreasing NP to binder ratio to a point where it is not
feasible for coating. Hence, the reported compositions in Table 5.1 were selected to
achieve feasible viscous suspensions for coating. Suspensions for coating were prepared
by mixing TiO2 NPs with binders A, B, and C at 1:4 to 1:16 (TiO2: Binder) weight ratios
in a mortar for about 15 min. Stainless steel coupons were then dip coated with the
resultant suspensions as described earlier. The coated coupons were air-dried over night
at room temperature. These coated coupons were found to be uniform and physically
148
stable upon scratching with pencil points of 2H hardness. Also, washing under running
water as well as sonication in water bath for about 15 min did not remove the coating
from the substrate significantly. Hence, TiO2 coatings with the binders A, B, and C were
selected for further studies to evaluate their physical stability.
Surface characteristics of TiO2 nanocoatings
In order to maintain consistency in use of samples for evaluating both physical
stability and bactericidal properties of the nanocoatings, an indented stainless steel
coupon having dimensions 46 x 12.5 x 1.25 mm3 and surface area of 540 mm
2 were used
for the TiO2 coatings with the binders A, B, and C, respectively. A sample of 0.25 g of
the coating solution at 1: 4 to 1:16 NP to binder weight ratio were poured into the well of
stainless steel indentation to form a uniform layer of TiO2 coating. The coated coupons
were dried in air overnight at room temperature. This approach of coating is referred as
solution deposition technique. The thickness of the coatings was measured by using a
thickness gauge (Elcometer® 345) at eight different locations on each coupon. Film
morphology and microscopic structure of the coating surface was characterized by a
variable pressure scanning electron microscope (VPSEM, Zeiss 1450 EP) with
accelerating 25 kV. The SEM images were further analyzed using an image processing
software (Paint. NET) to estimate the area ratio of coated surface covered by the NPs vs
the binder.
Measurement of physical stability of TiO2 nanocoatings
The hardness of the coatings was evaluated with the help of a scratch test based
on the ASTM G171-03 method (ASTM, 2009). Briefly, Instron fitted with a
hemispherical diamond tip indenter of 76.5 µm having a conical apex angle of 120°
149
(J&M Diamond Tool, Inc., Rumford, RI, USA) and an anti-vibration table was used for
this test. Three linear scratches of at least 5 mm length at 2 mm apart from each other
were made on each coating with an applied load of 1N to 3N. The width of each scratch
was measured at three different locations equidistance from each other using a digital
microscope pro (Celestron LLC, Model # 44308). Scratch hardness number (HSp) was
calculated as per the standard using following equation:
HSp = kP/w2
Where
HSp is the scratch hardness number (GPa)
K is the geometrical constant (24.98)
P is the applied normal force (grams-force)
W is the scratch width (µm)
In addition, adhesive strength of the coatings was evaluated using a scotch tape
test based on the ASTM D3359-02 test method B (ASTM, 2002). Six parallel cuts of
about 20 mm length at 2 mm apart were made through the coating in one steady motion
using a straight edged metal guide and a sharp razor blade as described in ASTM D3359-
02. Similarly, another six cuts were made through the coating at a 90̊ angle to the
previous cuts to make a lattice pattern of small squares of about 0.5 x 0.5 mm2
dimensions on the coating. Later, about 25 x 50 mm2 pressure-sensitive adhesive tape
(Permacel 99, Permacel, New Brinswick, NJ, USA) was applied over the lattice pattern
and smoothed into the place by using a pencil eraser over the area of the incisions to
ensure good contact with the coating. Adhesive tape was then removed by pulling it off
rapidly with a constant force at close to a 180° angle. The possible crumbling at the edges
150
of the cuts is a measure of the coating adhesion strength and is ranked from 5B to 0B
according to the descriptions and illustrations provided in the ASTM standard (Table
5.2).
A cleaning procedure commonly used in food processing environment was simulated
by means of an in-house developed reciprocating test (Fig 5.4). In this test set-up, a
texture analyzer (TA. XT Plus™
, Texture Technologies Corp, Scarsdale, NY, USA) was
fitted with a moving head consisting of scrubby side of sponge (3M Scotch-Brite™
,
Heavy-Duty scrub sponge, St. Paul, MN, USA) as four individual brushes to simulate
cleaning procedure (Fig 5.4b). Briefly, the weight of each coated coupon was measured
using a calibrated balance before subjecting to cleaning procedure. Later, a 2 mL of
diluted detergent solution (Dawn Ultra™, Procter & Gamble, Cincinnati, OH, USA) was
poured onto each TiO2 coated coupon. The coupons were then subjected to cleaning
procedure using the test set-up described earlier at a moving head speed of 20 mm/s for
up to 500 cycles (i.e. 1000 to-and-fro motions) with an applied load of 1 N on each
coupon. The cleaned coupons then were air-dried overnight at room temperature and the
weight of each coupon was measured again. The difference in the weights of TiO2 coated
coupons before and after cleaning was measured to determine the wear resistance of the
TiO2 coatings.
Measurement of bactericidal property of TiO2 nanocoatings
In order to select most appropriate test method to determine photocatalytic
bactericidal activity TiO2 nanocoatings, three different techniques: (i) Direct spreading,
(ii) Glass cover-slip, and (iii) Indented coupons were investigated. TiO2 coating with
binder A on stainless steel coupons (25 mm2) were used in direct spreading and glass
151
cover-slip techniques; while indented stainless steel coupons (540 mm2) as described
earlier were used for the indented coupon method. Further, E.coli has been widely studied
bacteria in several of the photocatalytic disinfection experiments involving TiO2 NPs.
However, the susceptibility of pathogenic strains of E.coli to photocatalytic disinfection
is not well reported. As a reason, a five strain cocktail of E. coli O157: H7 isolated from
different sources: E009 (beef), EO932 (cattle), O157-1 (beef), O157-4 (human), and
O157-5 (human) was used as a test pathogen in this study. Each bacterial stain was
cultured separately in 10 mL of tryptic soy broth (TSB) (Difco, Becton Dickinson,
Sparks, MD, USA) and kept on a shaking incubator at 230 rpm and 37°C for 16 h.
Following the incubation, bacterial cells were harvested by sedimentation at 4000 x g for
12 min and re-suspended in a sterile phosphate-buffered saline (PBS, pH 7.2). An equal
volume (2 mL) of each strain suspension was combined to obtain a 10 mL of a five strain
cocktail containing approximately 107 CFU/mL bacterial cells. Cell concentration was
adjusted by measuring the absorbance of bacterial suspension at 600 nm using a UV/Vis
spectrophotometer and confirmed by plating 100 µL portions of the appropriate serial
dilution on tryptic soy agar (TSA) (Difco Laboratories) plates incubated at 37 °C for 24
h.
Prior to antibacterial activity tests, TiO2 coated coupons were pre-sterilized under
UVC light for 1 h in a bio-safety cabinet. The sterile coupons were placed in a 90 mm
diameter petri-dish containing moist filter paper to maintain humidity during the
treatment. Bacterial culture was inoculated on each TiO2 coupon as follows: (i) Direct
spreading method: A drop of 100 µL inoculum was spread on the surface of TiO2 coating
using a sterile loop based on the direct spreading technique, (ii) Glass cover-slip method:
152
A drop of 100 µL inoculum was spread on the surface of TiO2 coating as before and then
a glass cover-slip of same size as stainless steel coupon was placed on top of the bacterial
culture, (iii) Indented coupon method: A 300 µL inoculum was pipetted into the well of
indented TiO2 coated coupon to cover entire indented coated surface. The samples were
then illuminated from above with a UV-A light system (American DJ, Model UV Panel
HP ™, LL-UV P40, Los Angeles, CA, USA) at 2 mW/cm2 intensity. The intensity of
light reaching the surface of the coating was measured using a UV radiometer (UVP®,
Upland, CA, USA). Plain stainless and only binder coated stainless steel coupon under
UV-A light were used as negative and positive controls. After 2 h UV treatment, TiO2
coated coupons were immersed in 10 mL or 30 mL (for indented coupon technique) PBS
solution containing 0.1% tween 80 and vortexed for 30 s to re-suspend the bacteria. A
viability count was performed by appropriate dilution and plating on E.coli O157:H7
selective Sorbitol MacConkey agar (SMAC) and incubation at 37°C for 24 h. All the
experiments were conducted in triplicate.
Statistical analysis
Data were analyzed by the analysis of variance (ANOVA) procedure using Statistical
Analysis System (SAS/STAT 9.3, 2011). T-tests were used for pairwise comparisons.
Least significant difference of means tests was done for multiple comparisons, and all
tests were performed with a level of significance 0.05.
Results and Discussion
Effect of binders on TiO2 coatings
Fig. 5.1 shows the images of different TiO2 coatings with binders A, B, and C
prepared by suspension deposition technique. As seen in the figure, all the nanocoatings
153
were uniform and strongly adhered to the stainless steel substrate. We found that
increasing the binder concentration in the coating resulted in smoother surfaces with
fewer visible aggregates as seen in the image of sample TB16 when compared with
sample TB8. Also, no cracks were formed on the TiO2 coating with binders A, B, and C.
However, TiO2 coating with binder A at 1:4 NP to binder weight ratio (i.e., TA4) showed
formation of cracks along the edges of the coating. As the concentration of binder A
further increased (TA8), no visible cracks were noticed on the coating. Thickness of TiO2
coatings with binders A, B, and C at different NP to binder weight ratios were shown in
Table 5.3. Thickness of nanocoatings ranged from 50 to 107 µm. In general, increasing
the concentration of binder in the coating decreased the thickness of nanocoatings. At the
same NP to binder composition (for example at 1:8 NP to binder weight ratio), thickness
of TiO2 coating with binder C (97 µm) was found to be significantly higher than the
thickness of TiO2 coatings with binders A (74 µm) and B (51 µm). The difference in the
thickness of these nanocoatings may be attributed to the differences in the viscosity of
coating solutions formulated using different binding agents and the relative proportion of
NPs to binder concentration in each type of nanocoating. Li and others (2009) reported
that the thickness of TiO2 membranes developed with PVA binder on stainless steel
decreased with decreasing the molar concentration of TiO2 in the casting solution. They
found that the thickness and micro-pores of the coatings can be controlled by simply
adjusting the concentration of casting solutions instead of applying multiple coats.
Similar results were reported by Cerna and others (2011) for TiO2 coated layers with
varying levels of PEG. Since photocatalytic antimicrobial activity of TiO2 is a surface
dependent phenomenon due to generation of ROS, the thickness of the coating becomes
154
insignificant. However, thickness of the coating may have a significant effect on the
physical stability and other structural properties of the TiO2 coatings.
Surface characteristics of TiO2 coatings
Fig. 5.2 shows scanning electron micrographs of surface of different TiO2
coatings. SEM images give us a detailed look at appearance of the deposited coatings at a
micro level. At the same level of NP to binder concentration, TiO2 coatings with binders
A, B, and C have shown completely different structural characteristics as seen in Fig. 5.2.
TiO2 coating with binder A (TA8) is more compact in nature with aggregated clumps on
the surface (Fig 5.2b). While, the surface of TiO2 coating with binder B (TB8) is compact
with several microscopic pores throughout the coating (Fig 5.2c). Whereas, TiO2 coating
with binder C (TC8) resulted in a compact structure without aggregated clumps with
larger but fewer number of pores on the surface (Fig 5.2e). Inset of the respective SEM
images of TA8, TB8, and TC8 shows the structural arrangement of the TiO2 coatings at
nanoscale. Upon analyzing these SEM micrographs at nanoscale to estimate the surface
coverage of NP vs binder showed a 40, 21, and 39 % coverage of binder and 60, 79, and
61% coverage of TiO2 NPs for coatings TA8, TB8, and TC8, respectively (Fig 5.2).
However, the actual number of TiO2 NPs that are exposed on the surface of the coating
were just a fraction of total percent coverage of TiO2 NPs. For example in Fig 5.3, if we
analyze the magnified image of TiO2 coating with binder C (TC8) at nanoscale; about 39
% of the coating surface was covered with the binder (dark black region), 58 % of the
surface was covered by the unexposed TiO2 NPs which are partly shielded by the binder
particles (blurred grey region), and rest of the 3 % of the surface was covered by the
exposed TiO2 NPs (bright white spots).
155
Further increasing the concentrations of binders B and C in the coating (i.e.
TB16 and TC16) resulted in a more compact surface structure with fewer number of
pores as shown in Figs. 5.2(d), and 5.2(f). This shows that the type and the amount of
binder used in the coating has a significant effect on the morphological and structural
properties of the TiO2 coating. This phenomenon is more obvious when ready-made TiO2
NPs were mixed with different binding agents for coating. Also, it is expected to generate
some irregularities and non-uniformity in the surface of coating while using the solution
deposition technique in the indented stainless steel coupon. However, the results of this
study help to prove the concept of developing durable antimicrobial nanocoatings on food
contact surfaces using appropriate binding agents.
Physical stability of TiO2 coatings
Different test procedures were adopted in order to estimate the physical stability
of the TiO2 coatings for use in food processing environment. Adhesion strength of the
TiO2 coatings was determined by following ASTM D3359-02 standard and the results
were reported in Table 5.3. TiO2 coatings with binder B (TB8 or TB16) showed the
highest adhesion rating of 4B. Here, rating 4B indicates that less than 5 % of the coating
has been removed from the surface as represented in Table 5.2. On the other hand, TiO2
coating with binder C at 1:16 NP to binder weight ratio (TC16) showed the lowest
adhesion strength of 2B which means more than 65% of the coating has been delaminated
from the surface. Further, increasing the concentration of NPs in the coating formulation
to 1:8 NP to binder weight ratio (TC8) significantly enhanced the adhesion strength of the
coating up to 4B. A similar trend was observed for TiO2 coating with binder A (TA8 or
TA4). This indicates that depending upon the type of binder, there exists an optimum
156
concentration of NPs and binder in the coating in order to achieve highest adhesion to the
substrate. For the tested binding agents in this study, a coating suspension at a
concentration of 1:8 NP to binder weight ratio was found to be the optimum for
exhibiting the highest adhesion strength.
The hardness of the coating was determined as per ASTM G171-03 standard
using a scratch resistance test. Preliminary experiments were conducted to determine the
maximum normal force that can be applied on the surface of TiO2 coating and a normal
force of 2 N was found to be the optimum to determine and compare the scratch
resistance of different TiO2 coatings developed in this study. Control samples with only
binder coating failed to withstand the scratch resistance test. Scratch hardness of TiO2
coatings ranged from 0.14 GPa for sample TA8 to 1.08 GPa for sample TB8 (Table 5.3).
TiO2 coatings with binder B showed highest scratch hardness followed by coatings with
binder C and A, respectively. Scratch resistance of the TiO2 coatings developed in this
study using different binders were found to be comparatively much higher than the
chemical vapor deposited TiO2 coatings on stainless steel substrate which was 6.5 GPa at
40 mN (Sobczyk-Guzenda and others 2013) and sol-gel dip coated TiO2 coatings on
polycarbonate sheets which was 0.5±0.04 GPa at 25 µN (Yaghoubi and others 2010).
Wear resistance of the TiO2 nanocoatings after simulated cleaning procedure was
reported in Table 5.3. The weight loss (mg) after 1000 cycles of simulated cleaning
procedure was expressed as wear resistance. The weight loss of the TiO2 nanocoatings
after wear testing ranged from 1.53 (for TC16) to 14 (for TB8) mg. TiO2 coatings with
binder C had the highest wear resistance (less weight loss) followed by TiO2 coatings
with binders A and B, respectively. In general, increasing the binder concentration in the
157
coating increased the wear resistance. However, the difference is not statistically
significant (p<0.05) except for TB8. In addition, subjective analysis of the coatings after
wear testing revealed that all the TiO2 nanocoatings looked physically very stable with
slight scratch marks on the surface. However, after the wear test was followed by drying,
formation of cracks and peeling-off of the coatings from the substrate was noticed for
TB8. This might be attributed to the weak intermolecular bonds between the NPs and the
binder at 1:8 NP to binder weight ratio for TB8.
Effect of test method on bactericidal activity results of TiO2 coatings
Shellac (binder A) is a food-grade natural resin most commonly used in the food
industry for several applications. Also, based on the structural characteristics of the TiO2
coatings with binder A, no significant difference in the total coverage of TiO2 NPs was
observed with decreasing NP concentration in the coating (Fig. 5.2). In addition, these
coatings exhibited good physical stability on stainless steel surface. For this reason, TiO2
coating with binder A was selected as a representative nanocoating to identify a suitable
testing method to determine the bactericidal property. The most widely reported direct
spreading and glass cover-slip techniques were compared with an indented coupon
technique developed in this study. The results of antimicrobial activity of TiO2 coatings
are shown in Table 5.4. Under tested conditions, negative control samples (plain stainless
steel coupons) and positive controls (binder coated stainless steel coupons) under UV-A
light showed a reduction in between 1.5 to 2.5 log CFU/cm2. No significant difference
(p>0.05) in the reduction among control samples was observed for direct spreading, and
indented coupon techniques. This shows that the binder coating itself (based on positive
control results) had no significant antimicrobial property and the observed reduction was
158
only be attributed to the effect of UV-A light. However, a significant difference in the
reduction between the two different control samples was observed in case of glass cover-
slip technique. Also, it should be noted that there was no significant difference (p >0.05)
in reduction between positive control and the TiO2 coatings at different NP
concentrations for the glass cover-slip technique. For the other two methods, TiO2
coatings showed significantly higher microbial reductions when compared with control
samples. This shows that the glass cover-slip technique may not be suitable for the
determination of bactericidal property of TiO2 nanocoatings, especially when TiO2
coatings were created using a binder. This can be explained based on two possible
reasons: i) A cover slip on the inoculated coupon helps to achieve uniform coverage of
bacterial cells on the surface of the nanocoating. However, it also inhibits the presence of
catalyst such as atmospheric oxygen which otherwise plays an important role in the
heterogeneous photocatalysis involving TiO2 to generate ROS, ii) The amount of surface
occupied NPs were limited when nanocoatings were prepared by mixing with a binding
agent as explained in the surface characteristics of nanocoatings in this study (Fig. 5.2).
In such a case, a cover-slip on the inoculated nanocoating promotes only localized
reactions on the coated surface and reduces the efficacy of photocatalytic bactericidal
property of the nanocoating. In addition, leakage of inoculated bacterial culture from the
sides of the coupon is difficult to avoid by using a cover-slip technique. Similar,
concerns has been expressed by Mills et al (2012) and they suggested using an alternative
approach such as a simple well system into which a standard volume of the bacterial
suspension is applied to the sample which lies at the bottom of the well.
159
Increasing the concentration of NPs in the TiO2 coating from 1:16 to 1:8 NP to
binder weight ratio did not significantly increase in the reduction observed using the
direct spreading technique. As per the SEM image analysis results of this study, it is
expected to achieve higher log reduction for coatings with more NPs due to more surface
coverage by TiO2 NPs. However, this did not happened using the direct spreading
technique. This may be due to non-uniform coverage of the inoculum on the entire
surface of TiO2 coating when using direct spreading technique. Whereas, the indented
coupon technique showed a significant increase (p<0.05) in the microbial reduction by
increasing the concentration of NPs in the coating (TA16 vs TA8). Even though there is
was no significant difference in the reduction within the same sample among the three
tested techniques, the indented coupon technique helped to achieve more consistent
results by minimizing variations in the determination of TiO2 antimicrobial property. A
similar technique has been used by Cushnie et al (2010). Mills et al (2012) reported that
the advantage of this type of approach is that it allows the bacterial suspension to be
accurately deployed to a known area of surface under investigation. As per our
observation the major benefits of using the indented coupon technique are: i) to achieve
uniform coverage of inoculated bacterial cells on the entire surface of the coating, ii) to
minimize the sample to sample variation and hence decreases the standard deviation, iii)
to achieve more available surface area, and iv) to allow the presence of oxygen for
efficient photocatalytic disinfection to takes place. Under tested conditions, the results of
current study suggest that the indented coupon technique is a more appropriate method to
determine bactericidal efficacy of photocatalytic TiO2 coatings.
160
Conclusions
This study has identified three promising binding agents to develop physically
stable TiO2 coatings on food contact surfaces. Image analysis of the coated surfaces
revealed that increasing the binder concentration in the coating decreased the exposed
TiO2 NPs on the surface which may reduce the bactericidal property of TiO2 coatings.
TiO2 coatings with polyurethane as a binder showed the highest scratch resistance
followed by coating with polycyclic and shellac, respectively. TiO2 coatings with
polyurethane and polycrylic at 1:8 NP to binder weight ratio showed the highest adhesion
to the substrate. Overall, TiO2 coating with polycrylic showed the highest physical
stability followed by nanocoating with polyurethane and shellac. An indented coupon
technique was found to be the most appropriate to test the bactericidal property of TiO2
coatings. Follow up studies need to be conducted to determine optimum conditions to
exhibit the highest bactericidal property by the developed TiO2 coatings under repeated
use conditions.
Acknowledgments
Funding for this study was provided by Agriculture and Food Research Initiative
grant no 2011-68003-30012 from the USDA National Institute of Food and Agriculture,
Food Safety: Food Processing Technologies to Destroy Food-borne Pathogens Program-
(A4131).
Author Contributions
Authors V. K. Yemmireddy and Yen-Con Hung designed experiments and wrote
the manuscript. V. K. Yemmireddy performed all the experiments and conducted data
161
analysis. Author Glenn D. Farrell provided technical assistance in designing and
fabricating instruments for physical stability assessment.
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Table 5.1. Composition of different TiO2 nanocoatings
1TiO2 nanocoatings with binders A, B, and C.
2Binders A, B, and C are shellac, polyurethane, and polycyclic, respectively.
3Composition of the coating suspension.
Sample code1 Type of binder
2 Composition
3 (weight basis)
TiO2 NPs Binder
TA4 A 1 4
TA8 A 1 8
TB8 B 1 8
TB16 B 1 16
TC8 C 1 8
TC16 C 1 16
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Table 5.2. ASTM D3359-02 classification of adhesion test results
166
Table 5.3. Physical stability results of TiO2 nanocoatings with different binders
Coating type1 Thickness (µm)
Hardness2(GPa) Adhesion rating
3
Wear resistance4 (mg)
TA4 107 ±17.34a
0.15±0.02d
3B 5.53±0.86b
TA8 74 ±11.67c
0.14±0.11d
3B 3.47±1.94b
TB8 51 ±8.09c
1.08±0.25a
4B 14.0±2.03a
TB16 50 ±7.04c
0.88±0.11ab
4B 5.18±2.87b
TC8 97 ±2.35ab
0.68±0.08bc
4B 1.67±0.83b
TC16 56 ±8.98c
0.55±0.06c
2B 1.53±0.29b
Mean values with the same superscript in the same column are not significantly different
(p >0.05). 1TiO2 coatings with binders A, B, and C at 1:4 to 1:16 NP to binder weight ratio.
2Scratch hardness number at 2 N based on ASTM G171-03 method.
3Adhesion rating (5B: Superior; 0B: Inferior) based on ASTM D3359-02 method-B.
4Weight loss in mg after subjecting to simulated washing procedure.
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Table 5.4. Bactericidal activity of TiO2 nanocoatings using different test
methods
Treatment
Log reduction (CFU/cm2) at 2 mW/cm
2 for 2 h
Direct
spreading
Glass
cover-slip
Indented
coupon
Negative control1
2.42±0.04b
1.47±0.27b
2.19±0.10c
Positive control2
2.45±0.39b
2.15±0.32a
2.31±0.15c
TA16 3.31±0.48a
2.96±0.64a
3.04±0.07b
TA8 3.15±0.47a
2.76±0.60a
3.57±0.48a
Mean values with the same superscript in the same column are not significantly
different (p >0.05). 1Plain stainless steel coupon under UVA.
2Only binder A coated coupon under UVA.
3TiO2 coating with binder A at 1:16 NP to binder weight ratio.
4TiO2 coating with binder A at 1:8 NP to binder weight ratio.
168
Fig 5.1. Images of TiO2 nanocoatings with shellac (A), polyurethane (B), and polycrylic
(C) binders at different NP to binder concentrations. Where, TA4 (TiO2 coating with
binder A at 1:4 NP to binder weight ratio), TA8, TB8, TC8 (TiO2 coating with binder A,
B, and C at 1:8 NP to binder weight ratios), TB16, TC16 (TiO2 coating with binder B and
C at 1:16 NP to binder weight ratios).
169
Fig
5.2
. S
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of
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surf
ace
of
TiO
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ings
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at
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.2a.
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.2b, 5.2
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.2e
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TiO
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oat
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ith b
inder
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TA
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TB
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an
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TC
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at 1
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P t
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inder
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.2d,
and 5
.2f
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TiO
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oat
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inder
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TB
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), a
nd C
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16)
at 1
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gh
t ra
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res
pec
tivel
y.
170
Fig 5.3. SEM image of TiO2 coating with binder C at 1:8 NP to binder weight ratio (TC8)
showing regions of binder, surface exposed TiO2 NPs, and unexposed TiO2 NPs that are
partly covered by the binder.
171
Fig 5.4. In-house fabricated wear resistance tester (a) Cleaning heads fitted to a texture
analyzer to perform reciprocating motion (b) Enlarged image of cleaning heads and
platform to fit coated coupons in place.
172
CHAPTER 6
EFFECT OF BINDER ON THE PHYSICAL STABILITY AND BACTERICIDAL
PROPERTY OF TITANIUM DIOXIDE (TIO2) NANOCOATINGS ON FOOD
CONTACT SURFACES4
4Veerachandra K. Yemmireddy and Yen-con Hung. Food Control (2015) doi:
10.1016/j.foodcont.2015.04.009. Reprinted here with permission of the publisher.
173
Abstract
TiO2 is a promising photocatalyst for use in food processing environment as an
antimicrobial coating. The purpose of this study was to determine the effect of different
binding agents on the physical stability and bactericidal property of TiO2 nanocoatings
created on stainless steel surfaces. A total of six different coating suspensions were
prepared by mixing TiO2 (Aeroxide®
P-25) nanoparticles (NPs) with three different types
of binders (Shellac (A), polyuretahne (B), and polycrylic (C)) at a 1:4 to 1:16 NP to
binder weight ratio. Bactericidal activity of these TiO2 coatings against Escherichia coli
O157:H7 (5-strain) was determined at three different UV-A light intensities (0.25, 0.50
and 0.75 mW/cm2) for 3 h. The type of binder used in the coating had a significant effect
on the log reduction of E.coli O157:H7. TiO2 coatings with binder C showed highest
reduction (> 4 log CFU/cm2) followed by TiO2 coating with binder B and A. Increasing
the binder concentration in the formulation from a 1:4 to 1:16 weight ratio decreased the
log reduction of E.coli O157:H7. Increasing the UV-A light intensity from 0.25 to 0.75
mW/cm2 increased the log reduction of bacteria for all the TiO2 coatings. The physical
stability of the TiO2 coatings was determined using ASTM procedures. TiO2 coatings
with binder B showed highest adhesion strength and scratch hardness when compared to
coatings with other binders. However, on repeated use experiments (1, 3, 5, and 10
times), TiO2 coatings with binder C were found to be physically more stable and able to
retain their original bactericidal property. The results of this study showed promise in
developing durable TiO2 coatings with strong photocatalytic bactericidal property on
food contact surfaces using appropriate binding agents to help ensure safe food
processing environment.
174
Keywords: TiO2; Antimicrobial coating; Physical stability; Binders; E. coli O157:H7.
Highlights:
Most efficient TiO2 coating achieved more than 4 log reduction of E.coli
O157:H7.
Type of binder used in the coating has a significant effect on the log reduction.
Increasing the UVA intensity increased the bactericidal efficacy of TiO2 coatings.
TiO2 coating with polycrylic as binder showed the highest physical stability.
175
1. Introduction
Titanium dioxide (TiO2) is a well-known photocatalyst with excellent
antimicrobial properties under UV-A light. It is widely utilized as a self-cleaning and
self-sterilizing material for surface coatings in many applications (Fujishima, 2000). TiO2
is stable, non-toxic, cheap, and capable of repeated use without substantial loss of
catalytic ability. TiO2 photocatalysts have been added to paints, cements, windows, tiles
or other building products due to its sterilizing and anti-fouling properties (Lan et al.,
2013). Decontamination occurs under ambient conditions utilizing natural oxygen
without forming any photo-induced intermediates (Chong et al, 2010). In addition, TiO2
has been approved by the American Food and Drug Administration (FDA) for use in
human food, drugs, cosmetics, and food contact materials (Maneerat & Hayata, 2006).
Since Matsunaga et al. (1985) reported the application of photocatalysis for the
destruction of Lactobacillus acidophilus, Saccharomyces cerevisiae, and Escherichia coli
using platinum-loaded TiO2, there has been increased interest in the biological
applications of this process. TiO2 photocatalysts have been studied extensively to
inactivate a broad spectrum of microorganisms including viruses, bacteria, fungi, and
algae as well as to kill cancer cells (Kim et al., 2003). Foster et al. (2011) presented a
more comprehensive review on photocatalytic antimicrobial properties of TiO2. TiO2
photocatalysts generate strong oxidizing power when illuminated with UV-A light of
wavelength less than 385 nm. The bactericidal properties of TiO2 are attributed to the
high redox potential of the reactive oxygen species (ROS) such as hydroxyl radical (.OH),
superoxide radical (O2.-), and hydrogen peroxide (H2O2) formed by the photo-excitation.
TiO2-mediated photo-oxidation shows promise for the elimination of microorganisms in
176
areas where the use of chemical cleaning agents or biocides is ineffective or is restricted
by regulations such as pharmaceutical and food industries (Skorb et al., 2008). In
addition, TiO2 becomes superhydrophilic upon irradiation with UV light and this
functionality is reversible and depends on the light exposure (Chen & Mao, 2007). These
properties of TiO2 may help to improve the efficiency of hydrophilic cleaning agents
used in the food industry. Thus, TiO2 photocatalysts offer great potential to develop
antimicrobial coatings on food contact and non-food contact surfaces to avoid cross-
contamination in the food processing environment.
Studies have reported that immobilized TiO2 coatings have the ability to disinfect
Listeria monocytogenes biofilms on stainless steel (Chorianopoulos et al., 2011). Also,
TiO2 coated polypropylene film package can reduce the growth of E. coli on cut lettuce
(Chawengkijwanich & Hayata, 2008), and Pencillium expansum fruit rot on apples and
tomatoes (Manreet & Hayata, 2006). However, most of the earlier studies that reported
antimicrobial activity of TiO2 nanocoatings either used complicated approaches for
coating or did not fully address the issues of durability of the coatings on usage. In our
previous study on nanocoatings, we developed a simple method to create physically
stable TiO2 coatings on stainless steel surfaces using shellac, polyurethane and polycrylic
as binding agents (Yemmireddy et al., 2015). For developing antimicrobial TiO2
nanocoatings on food contact surfaces for the purpose of maintaining a hygienic food
processing environment, the binding agents used must be non-toxic. Shellac is an insect-
produced natural resin most commonly used in food industry for surface
treatment/glazing of confectionary products and citrus fruits to prevent surface damage
during handling and storage (Antic et al., 2010). According to FDA, shellac is only
177
approved for indirect food contact use (21 CFR 175.300). However, it is allowed for food
contact use due to acceptance petition for GRAS status (Baldwin, 2005). Shellac films
show excellent adhesion to a wide variety of surfaces and possess high gloss, hardness
and strength. Alternatively, polyurethane has been extensively studied for several
industrial applications. Notably, waterborne polyurethanes are suitable for paints,
coatings, and adhesive industries due to their inherent advantages of low volatile
compounds, fast drying properties, outstanding flexibility, impact resistance, abrasion
resistance, non-flammability, transparency and easy adherence to a variety of substrates
(Bhargava et al., 2013). As per FDA (21 CFR 177.1680), polyurethane resins are allowed
to use as indirect food additives for use as basic components of single and repeated use
food contact surfaces. Similarly, polycrylics are well known for their wide range of
applications in several paint formulations. As per our earlier study, TiO2 coatings created
using these binders have shown excellent physical stability. However, the photocatalytic
bactericidal property of TiO2 coatings using these binders is not well understood. Hence,
the overall objective of this study was to determine the effect of different binding agents
on physical stability and bactericidal property of TiO2 nanocoatings. Specific objectives
include: To determine:
i) The effect of binder on bactericidal property of TiO2 nanocoatings.
ii) The optimum conditions to create TiO2 nanocoatings with strong bactericidal
property
iii) The durability and bactericidal property of TiO2 nanocoatings on repeated use.
178
2. Materials and methods
2.1 Selection of materials
TiO2 (Aeroxide® P25, Sigma-Aldrich, St. Louis, MO, USA) NPs with an
approximate particle size of 21 nm and specific surface area of 50 m2 g
-1 as per suppliers
specifications were used for developing nanocoatings in this study (Table 1). Three
different binders namely, shellac (A), polyurethane (B) and polycrylic (C) were
purchased from the local supermarket in Griffin, GA (Table 1). Stainless steel (AISI
304L) coupons having an indentation with 46 x 12.5 x 1.25 mm3 dimensions and a total
surface area of 540 mm2 were chosen as a model food contact surface for TiO2
nanocoating. All the coupons were thoroughly cleaned prior to coating first by washing in
acetone followed by ethanol and finally rinsed with deionized water and dried in a hot air
oven at 60̊ C for 30 min.
2.2. Preparation of suspensions for TiO2 coating
Total six different suspensions of TiO2 were prepared by mixing TiO2 NPs with
binder A (1:4 or 1:8 weight ratio), binder B (1:8 or 1:16 weight ratio), and binder C (1:8
or 1:16 weight ratio) in a porcelain mortar for about 15 min. The produced viscous
suspensions were further treated in an ultrasonic water bath (Model # FS60, Fisher
Scientific, Waltham, MA, USA) for about 1 h, in order to avoid formation of TiO2
aggregates. The resultant viscous paste formulations were used for coating on indented
stainless steel coupons.
2.3. Preparation and characterization of TiO2 nanocoatings
TiO2 nanocoatings were created on indented stainless steel coupons by following the
method described in Yemmireddy et al. (2015). Briefly, a sample of 0.25 ± 0.02 g of
179
coating suspension was weighed into the well of an indented stainless steel (SS) coupon
by placing it on a calibrated balance. The deposited coating suspension was evenly spread
across the entire area of the indentation by slowly tilting the coupon sideways or if
needed using a Crayola paint brush by keeping total amount of deposited coating
constant. The coated coupons were air-dried over night at room temperature. The
resultant coatings has a thickness of about 50-100 µm when measured using a handheld
thickness gauge (Elcometer, Model # 345). The morphology and the microscopic
structure of the coating surface was characterized by a variable pressure scanning
electron microscope (VPSEM, Zeiss 1450 EP) with accelerating 25 kV. The SEM images
were further analyzed using image processing software (Paint. NET) to estimate the area
of the coated surface covered by the NPs and the binder.
2.4. Bacterial strains and inoculum preparation
Five strains of E. coli O157: H7 isolated from different sources: E009 (beef),
EO932 (cattle), O157-1 (beef), O157-4 (human), and O157-5 (human) were used in this
study. All bacterial strains were stored at -70 °C in tryptic soy broth (TSB) (Difco,
Becton Dickinson, Sparks, MD, USA) containing 20 % glycerol. Prior to the experiment,
cultures were activated at least twice by growing them overnight in 10 mL of TSB at 37
°C. Later, each bacterial strain was cultured separately in 10 mL of TSB and kept on a
shaking incubator at 230 rpm and 37°C for 16 h. Following the incubation, bacterial cells
were harvested by sedimenting at 4000 x g for 12 min and re-suspended in a sterile
phosphate-buffered saline (PBS, pH 7.2). An equal volume (2 mL) of each strain
suspension was combined to obtain a 10 mL of a five-strain cocktail containing
approximately 106 CFU/mL. Cell concentration was adjusted by measuring the
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absorbance of bacterial suspension at 600 nm using a UV/Vis spectrophotometer and
confirmed by plating 100 µL portions of the appropriate serial dilution on tryptic soy agar
(TSA) (Difco Laboratories) plates incubated at 37 °C for 24 h.
2.5. Photocatalytic disinfection
Prior to photocatalytic disinfection, the TiO2 coated coupons were pre-sterilized
under germicidal UV light (254 nm) in a biosafety hood for about 1 h. The sterilized
coupons were placed in 90 mm diameter petri-dishes containing moistened filter paper at
the bottom to prevent drying-out of the bacterial culture during the treatment. A 300 µL
aliquot of bacterial culture was pipetted into the indented well of the TiO2 coated coupon
and uniformly spread across the entire surface of the TiO2 coating using a sterile
disposable loop. Later, the inoculated samples were illuminated with a UV-A light
system fitted with four 40 W lamps (American DJ®, Model # UV Panel HP
TM, LL-UV
P40, Los Angeles, CA , USA) from above. The light intensity reaching on top of the
sample was measured using a UV radiometer (UVP®, Upland, CA, USA) with a peak
sensitivity of 365 nm. The light intensity reaching the surface of the sample was adjusted
to 0.25, 0.5 or 0.75 mW/cm2 (±0.05) by changing the distance between the light source
and the sample. Plain SS and only binder coated SS coupons under UV-A light were also
included as negative and positive controls, respectively. The samples were treated for
either 90 or 180 min UV-A light and then immersed in 30 mL of sterile PBS solution
containing 0.1% tween 80 and vortexed for 30 s to re-suspend the bacteria. A viability
count (log CFU/cm2) was performed by appropriate dilution and plating on E.coli
O157:H7 selective Sorbitol-MacConkey agar (SMAC) and incubation at 37 °C for 24 h.
All the experiments were conducted in triplicates.
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2.6. Measurement of coating physical stability
Hardness of the TiO2 coatings were assessed with the help of a scratch test, based
on ASTM G171-03 method (ASTM, 2009) as described in Yemmireddy et al. (2015) to
make a linear scratch of at least 5 mm length with an applied normal force of 2 N at three
different locations on each sample. The width of each scratch was measured at three
different locations equidistance from each other using a digital microscope pro (20 to
200x magnification, Model # 44308, Celestron LLC,Torrance, CA). Scratch hardness
number (HSp) was calculated as described in the standard and reported in Giga Pascals
(GPa). Further, adhesion strength of the coatings was determined with the help of a tape
test based on ASTM D3359-02 method-B (ASTM, 2002) as described in Yemmireddy et
al. (2015).
2.7. Simulation of repeated use conditions of TiO2 coatings
In order to determine whether the coatings were able to retain their original
bactericidal property and physical stability upon reuse, the coatings were subjected to
multiple use conditions. In this procedure, the coatings were subjected to photocatalytic
disinfection test conditions as described earlier such as pre-sterilization under germicidal
UV light for 1 h followed by photocatalytic disinfection treatment under UV-A light for 3
h and removal of bacterial cells from the coatings using release buffer for 30 sec were
simulated for 1, 3, 5, and 10 times using deionized water in place of actual bacterial
culture. After each treatment cycle the coupons were air-dried before proceeding to the
next cycle. Finally, the dried coupons after 1, 3, 5, and 10 times simulated use were
measured for their bactericidal property and the physical stability as described earlier.
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2.8. Statistical analysis
Data were analyzed by the analysis of variance (ANOVA) procedure using Statistical
Analysis System (SAS/STAT 9.3, 2011). T-tests were used for pairwise comparisons.
Least significant difference of means tests was done for multiple comparisons, and all
tests were performed with a level of significance 0.05.
3. Results and discussion
3.1. Effect of type and concentration of binder on the bactericidal activity of TiO2
nanocoatings
Fig. 1 shows the effect of type of binder on the log reduction of E.coli O157:H7
produced by TiO2 nanocoatings treated for 3 h at 0.5 mW/cm2 UV-A light intensity.
Control samples with plain stainless steel coupons, and only binder A, B, and C coated
coupons under UV-A light showed a reduction on E.coli O157:H7 population of only
0.17, 0.24, 0.51, and 2.23 log CFU/cm2, respectively. In addition, when these binder
coated coupons were tested in the dark, both binder A and B coatings showed no
significant antibacterial activity; while, binder C coating showed a reduction of less than
1 log CFU/cm2 (data not shown). This shows that under tested conditions, both binder A
and B coatings themselves had no significant bactericidal property. However, binder C
under the tested UVA intensity showed a significant (P ≤0.05) bacterial reduction. This
might be attributed to the possible inherent bactericidal properties of acrylic paint (i.e.
binder C) and its constituents in the presence or absence of UV light. TiO2 coatings with
binders A, B, and C at 1:8 NP to binder weight ratio showed a reduction of 0.96, 3.72,
and 3.92 log CFU/cm2, respectively (Fig 1). Further increasing the concentration of
binders A, B, and C in the TiO2 coating (1:16 NP to binder weight ratio) showed a
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reduction of only < 0.5 log CFU/cm2 for coating with binder A (TA16, data not shown),
1.73 log CFU/cm2 for binder B (TB16) and 3.35 log CFU/cm
2 for binder C (TC16) (Fig.
1). This is almost a 100, 54 and 15 % decrease in the bactericidal efficacy of TiO2
coatings with binders A, B and C when compared with respective TA8, TB8, and TC8
samples. Alternatively, decreasing the concentration of the binder in the TiO2 coating to
1:4 NP to binder weight ratio (TA4) resulted in an almost 49 % increase in the
bactericidal efficacy (0.95 to 1.45 log CFU/cm2) when compared to TiO2 coating at 1:8
NP to binder weight ratio (TA8). However, TiO2 nanocoatings with binders B and C at a
1:4 NP to binder weight ratio is not a feasible formulation for coating by the solution
deposition technique used in this study. This indicates that the type of binder used in the
TiO2 coating had significant (P ≤0.05) effect on the photocatalytic bactericidal property.
One possible reason for the differences in the antimicrobial activity can be attributed to
the differences in the surface characteristics of the individual TiO2 nanocoatings created
with three different binders.
SEM image analysis of the coatings revealed that the number of TiO2 NPs present
on the surface of each coating varied depending on the type of binder used (Table 2). For
example, in a given area of nanocoating, the amount of TiO2 NPs exposed on the surface
of coating was only 3, 2, and 3 % for TA8, TB8, and TC8, respectively. While, the
corresponding binder coverage was 39, 21, and 39 %, respectively. The remaining
percent coverage of the nanocoating can be attributed to the unexposed TiO2 NPs. The
unexposed TiO2 NPs are believed to be partly shielded by the binder molecules reducing
the ability of UV-A light penetration and bacterial cell contact with the TiO2 NPs. As per
our previous study, the type of binder used in the TiO2 coating has an effect on the
184
structural properties of the resultant coatings (Yemmireddy et al, 2015). SEM analysis of
the coatings revealed that the TiO2 coating with binder-A was more compact in nature
whereas the TiO2 coatings with binders B and C were porous in nature. The porous
structure of the TiO2 coatings with binders B and C might have helped to carryout
efficient oxidation and reduction reactions due to availability of electron donors (H2O)
and the acceptors (O2) from the immediate environment. This condition helps to generate
more ROS for photocatalytic disinfection of bacteria. Thus, the structural characteristics
of TiO2 coating and the number of TiO2 NPs that are directly in-contact with bacterial
cells during photocatalytic disinfection treatment plays an important role in the
generation of ROS responsible for the damage of cell walls and eventual cell death.
Many studies have reported that close contact between the bacteria and the TiO2
increases the extent of oxidative damage (Foster et al., 2011). This explains the reason for
the high bactericidal activity of TiO2 coatings with binder B and C when compared to
TiO2 coating with binder A. Based on these results it is clear that increasing the NP
concentration in the coatings increased the log reduction of bacteria. However, there
exists an optimum level of TiO2 to binder concentration to exhibit greater bactericidal
property depending upon the type of binder used in the coating. TiO2 coatings with
binder C showed the highest bactericidal activity followed by TiO2 coating with binder B
and binder A.
3.2.Effect of light intensity on the bactericidal activity of TiO2 nanocoatings
The effect of UV-A light intensity on the bactericidal activity of different TiO2
nanocoatings was shown in Fig 2. When UV-A intensity range from 0.25 to 0.75
mW/cm2, control samples with plain SS coupon showed a reduction of less than a 1 log
185
CFU/cm2 after 3 h treatment. In a similar experiments by Chawengkijwanich and Hayata
(2008), UV-A light itself showed a 1 log CFU/cm2 reduction of E.coli cells after 3 h
treatment at 1 mW/cm2. Similarly, Kikuchi et al. (1997) reported less than 2 log CFU/cm
2
reduction of E.coli cells after 4 h treatment at 1 mW/cm2. Another study by Krysa et al.
(2011), authors reported that increasing UV-A light intensity from 0.2 to 0.6 mW/cm2,
decreased the survival of E.coli cells from 77 to 38 % after a 3 h treatment. This can be
explained by the fact that UV-A light, with relatively low energy, gradually damages
cells through oxidative stress caused by generation of oxygen radicals within the cells
(Bock et al, 1998). The oxidative stress caused by UV-A light on bacterial cells might be
more pronounced with increasing light intensity and treatment time. This shows that UV-
A light itself has minimal bactericidal activity at low intensity levels used in this study.
Increasing the UV-A light intensity from 0.25 to 0.75 mW/cm2 also increased the
bactericidal activity of all TiO2 coatings (Fig 2). Coating with only binder A has showed
a reduction of 0.12, 0.24 and 1.27 log CFU/cm2 at 0.25, 0.5 and 0.75 mW/cm
2 UVA light
intensities, respectively. This indicates that the binder A coating itself has a negligible
effect on the reduction of bacteria at lower light intensities of below 0.50 mW/cm2 and
followed the reduction trend of the UV-A control. However, further increasing the light
intensity to 0.75 mW/cm2 increased the bactericidal activity of the binder coating
compared to the UVA control. Shellac (i.e. binder A) is a food-grade, insect produced
natural resin and widely used as a glazing agent in the food industry. The binder itself is
non-toxic and used in several other food applications. Antic et al. (2010) studied the
effect of shellac-in-ethanol solutions to reduce the transferability of bacteria from cattle
hide to the beef carcass during slaughter operation by immobilizing the bacterial cells on
186
the hide. They reported that shellac itself did not have significant antimicrobial effects
while shellac-in-ethanol showed some antibacterial effect. Similarly, a possible
synergistic effect between shellac under UV-A light at 0.75 mW/cm2 in the current study
might have resulted in a slightly increased reduction. Similarly, the binder B (i.e.
polyurethane) coating itself under UV-A light had little effect on bactericidal activity (Fig
2). Whereas, binder C (i.e. polycrylic) coating showed significantly (P≤0.05) higher
reduction from 2.5 to 4 log CFU/cm2 after a 180 min UV-A exposure (Fig 2).
Increasing the intensity of UV light from 0.25 to 0.75 mW/cm2 for 180 min,
increased the bactericidal activity of TiO2 coating from 0.63 to 1.69 log CFU/cm2 for
binder A (TA8) and from 2.45 to 3.87 log CFU/cm2 for binder B (TB8). However, no
significant (P>0.05) increase in the log reduction was observed for TiO2 coating using
binder C (TC8) (Fig 2). The minimum detection limit for the current test method is 2 log
CFU/cm2. It should be noted that TiO2 coatings with binder C (TC8) at 0.25 mW/cm
2
already reached the highest possible reductions (4 log CFU/cm2) for an initial bacterial
cell concentration of around 106 CFU/cm
2. This is why no additional reduction was
achieved for binder C (TC8) at a higher UV intensity. In order to determine UV intensity
effect, treatment times for TB8 and TC8 nanocoatings were reduced to 90 min (Fig 2).
This treatment step resulted in almost a 51% (for TB8) and 36 % (for TC8) decrease in
the bactericidal activity of TiO2 coatings with binder B and C when compared with
treatment for 180 min. This demonstrates that the observed reductions are in-fact due to
the pronounced photocatalytic bactericidal effect of TiO2 coatings. Marolt et al. (2011)
reported that the photocatalytic treatment on exposed anatase TiO2 nanoparticles could
result in a reactive species that would destroy the soft organic matter such as binders in
187
the vicinity of NPs, thus exposing even more anatase particles. Increasing the
concentration of NPs and UV-A light intensity might have destroyed and removed a
certain amount of the superficial binder and of the other degradable paint components
from the surface of coating thus increasing the bactericidal property of TiO2 nanocoating.
Chawengkijwanich and Hayata (2008) reported that increasing UV-A light intensity from
0.05 to 1 mW/cm2 increased the antimicrobial efficacy of TiO2 coated polypropylene
films from 0.35 to 3 log CFU/cm2. Similar results were also reported by Krysa et al.
(2011) and Dunlop et al. (2010). This indicates that the type of binder, the relative
proportion of the NP to the binder, and the intensity of UV light all have a significant
effect on the bactericidal property of TiO2 coatings. However, the photocatalytic activity
against pathogens at lower light intensity levels is more relevant to potential real life
applications (Foster et al., 2011). Hence extending the photocatalytic bactericidal
property of TiO2 coating towards lower UV-A light intensities or visible light region is
more beneficial. Based on the results of the current study, an UV-A light intensity of 0.5
mW/cm2 was found to be optimum for exhibiting bactericidal property of TiO2
nanocoatings.
3.3. Bactericidal activity of TiO2 nanocoatings on repeated use
Fig. 3 shows the bactericidal activity of TiO2 nanocoatings with binders A, B, and
C at 1:8 NP to binder weight ratio after the repeated use experiment. Except for the TiO2
coatings with binder B and C, there was no significant (P>0.05) loss of photocatalytic
bactericidal property of the TiO2 coatings with binder A was noticed after the multiple
use experiment. Originally, TiO2 coatings with binders A, B, and C (TA8, TB8, and TC8)
irradiated for 180 min at 0.5 mW/cm2 UVA light intensity exhibited a reduction of 0.96,
188
3.72, and 3.92 log CFU/cm2, respectively. However, after one time simulated use of
coated coupons, no significant difference in the reduction was observed for TiO2 coating
with binder A and the reduction remained around 1 log CFU/cm2 (TA8-1). Whereas,
TiO2 coatings with binders B (TB8-1) and C (TC8-1) had high initial log reduction but
lost almost 73 and 22 % of their original bactericidal property after one time use,
respectively. Further, testing the bactericidal property of TiO2 coatings with binder C for
the 3 (TC8-3), 5 (TC8-5) and 10 (TC8-10) times repeated use experiments did not show
significant further reduction in its bactericidal property. Upon repeated use, the change in
bactericidal efficacy of TiO2 nanocoatings can be attributed to the loss of exposed TiO2
NPs on the surface of coating. This is in part related to the decreased physical stability of
the respective coatings when subjected to the repeated use experimental conditions.
3.4. Physical stability of TiO2 nanocoatings on repeated use
Physical stability results of the TiO2 coatings with binder A (TA8), B (TB8), and
C (TC8) before and after subjecting to the repeated use experiment are shown in Table 3.
The thickness of all the TiO2 coatings decreased after the repeated use experiments. After
the one time use experiment, the thickness of coatings TA8, TB8, and TC8 decreased by
31, 29, and 12 %, respectively when compared with the thickness of original coatings.
Further subjecting the TiO2 coating with binder C (TC8) for 3, 5, and 10 times in the
repeated use experiment resulted in 38, 48, and 54 % decreases in the thickness of the
original coating. Adhesion strength of the TiO2 coatings was assessed based on ASTM
D3359-02 standard method –B. Originally, coatings TA8, TB8, and TC8 showed a mean
adhesion rating of 3B, 4B, and 4B, respectively. As per the ASTM standard, adhesion
strength is rated from 5B to 0B. Where, 5B means the coatings has superior adhesion
189
with 0% loss of coated area, followed by 4B (<5 %), 3B (5-15%), 2B (15-35%), 1B (35-
65%), and 0B (>65%), respectively. It means both the coatings TB8 and TC8 showed
good adhesion strength (4B) before subjecting to repeated use. After 1 time repeated use,
adhesion strength of TB8 decreased to 3B while no significant change in the adhesion
was observed for coatings TA8 and TC8 (Table 3). In addition, TC8 maintained the same
original adhesion strength (4B) even after subjecting for 5 times repeated use. However, a
decrease in the adhesion (from 4B to 3B) was noticed after the 10 times repeated use
experiment for TC8. This can be attributed to the corresponding decrease in the thickness
of the original coating from 97 µm to 45 µm after the 10 times repeated use experiment
as described earlier.
Scratch hardness of the TiO2 coatings with binders A, B, and C before and after
the reuse experiment was reported in Table 3. Originally, TiO2 coating with binder B
(TB8) showed the highest scratch resistance (1.08 GPa) followed by TC8 (0.68 GPa) and
TA8 (0.14 GPa), respectively. After the one time repeated use experiment, scratch
hardness of TB8 and TC8 were reduced to 0.61 GPa and 0.53 GPa, respectively.
Whereas, scratch hardness of TA8 increased to 0.42 after one time use. In a similar
manner, after the 3, 5, and 10 times repeated use experiments, the scratch hardness of
TC8 increased by 32, 32, and 13%, respectively when compared with original coating.
These differences in the scratch hardness among different coatings can be partly
attributed to the nature of the binders used in the coating. Depending on the nature of
binder used in the TiO2 coating the width of the scratch either increased or decreased
after the repeated use experiment. For example, the scratch width of the TiO2 coating
with binder A (TA8) increased from 240 µm to 112 µm after one time repeated use.
190
Since scratch width is inversely proportional to the scratch hardness number (as per the
ASTM standard), the scratch hardness of TA8 increased after the one time use
experiment. Whereas, the width of the scratch for TB8 (70 to 90 µm) and TC8 (90 to 100
µm) increased after one time use which led to a decreased scratch hardness number.
However, as the TiO2 coating with binder C (TC8) subjected for the 3, 5, and 10 times
repeated use experiments, the width of the scratch again decreased to 76, 77, and 82 µm
which resulted in an increase in scratch hardness of the coating.
Bhargava et al. (2013) studied the effect of TiO2 concentration (pigment-to-binder
ratio) and dispersing agent on the peel strength of waterborne-polyurethane based
coatings on aluminum substrates. They found that the adhesion strength of the coating
decreased with increasing pigment-to-binder ratio. This may explain the reason for the
decreased physical stability of TB8 after one time use in the current study. TiO2 coating
with binder B (polyurethane) at a 1:8 NP to binder weight ratio may not be sufficient to
impart high physical stability even though it exhibited good bactericidal property
originally. Kumar et al. (2012) reported that silicone functionalized TiO2 based epoxy
coatings on carbon steel exhibited higher values of scratch hardness, pull-off adhesion
and impact resistance. The synergistic interaction between pigment and polymer matrix
through chemical bonding is believed to be the reason for the high mechanical properties
of TiO2 based epoxy coatings. A similar interaction effect might be one possible reason
for the increased hardness of TA8 and TC8 even after repeated use. Based on these
results, adhesion strength and scratch hardness values of the coating were well correlated
with the retention of original bactericidal property of the TiO2 nanocoatings. Among the
tested nanocoatings, TiO2 coatings with binder C showed high bactericidal property and
191
physical stability after the repeated use experiment. These results indicate that type of
binder and the binder-to-nanoparticle concentration used in the coating has a significant
effect (P≤0.05) on the durability and bactericidal property of TiO2 coatings.
4. Conclusions
As per this study, TiO2 coatings with polycrylic as binding agent showed the highest
bactericidal efficacy followed by TiO2 coatings with polyurethane, and shellac as binding
agents, respectively. Increasing the concentration of binder in the TiO2 coating decreased
the bactericidal efficacy. Increasing the UV-A light intensity from 0.25 to 0.75 mW/cm2
increased the bactericidal activity of the TiO2 coatings. However, an intensity of 0.50
mW/cm2 was found to be optimum to avoid the effect of UV light itself on the bacterial
reduction. TiO2 coating with polyurethane as binding agent showed the highest adhesion
strength and scratch hardness. However, on repeated use experiments, TiO2 coating with
polycrylic was found to be physically more stable and bactericidal when compared with
other TiO2 coatings. The results of this study provide feasibility in development of
durable TiO2 nanocoatings with strong bactericidal properties on food contact surfaces
with appropriate binding agents.
Acknowledgements
Funding for this study was provided by Agriculture and Food Research Initiative
grant no 2011-68003-30012 from the USDA National Institute of Food and Agriculture,
Food Safety: Food Processing Technologies to Destroy Food-borne Pathogens Program-
(A4131). The authors would also like to thank Mr. Glenn Farrell for the technical
assistance.
192
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Table 6.1. Details of the binders and the composition of different TiO2 nanocoatings
Sample Description
TiO2 TiO2 Aeroxide® P25, surface area 50 m
2 g
-1 and particle size ~21 nm
Sigma-Aldrich, St. Louis, MO, USA
Binder A Shellac a natural resin
Zinsser Co., Inc. Somerset, NJ, USA
Binder B Water based oil modified polyurethane
Minwax®, Minwax company, Upper saddle river, NJ, USA
Binder C Water based polyacrylic
Minwax®, Minwax company, Upper saddle river, NJ, USA
TA4 Nanocoating with TiO2 and binder A at 1:4 weight ratio
TA8 Nanocoating with TiO2 and binder A at 1:8 weight ratio
TB8 Nanocoating with TiO2 and binder B at 1:8 weight ratio
TB16 Nanocoating with TiO2 and binder B at 1:16 weight ratio
TC8 Nanocoating with TiO2 and binder C at 1:8 weight ratio
TC16 Nanocoating with TiO2 and binder C at 1:16 weight ratio
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Table 6.2. Estimated surface coverage of the nanocoatings with the binder and the TiO2
nanoparticles
1TA4 is the TiO2 coating with binder A at 1:4 NP to binder weight ratio
TA8, TB8, and TC8 are the TiO2 coatings with binders A, B, and C at 1:8 NP to
binder weight ratio.
TB16, and TC16 are TiO2 coating with binder B and C at 1:16 NP to binder weight ratio.
Sample code1
Percent surface coverage based on SEM image
analysis (Estimate only)
Binder Exposed
TiO2
Unexposed
TiO2
Total
TiO2
TA4 38 8 54 62
TA8 39 3 58 61
TB8 21 2 77 79
TB16 33 5 62 67
TC8 39 3 58 61
TC16 43 2 55 57
197
Table 6.3. Physical stability of TiO2 coatings before and after repeated use
experiment
Coating
type1
No of times
used
Thickness (µm) Adhesion rating Hardness (GPa)
Before
After Before
After Before
After
TA8 1 74ab
51bcd
3B 3B 0.14f
0.42e
TB8 1 51bcd
36d
4B 3B 1.08a
0.61dce
TC8 1 97a
85a
4B 4B 0.68dc
0.53de
TC8 3 97a
60bc
4B 4B 0.68dc
0.90ba
TC8 5 97a
50cd
4B 4B 0.68dc
0.90ba
TC8 10 97a
45cd
4B 3B 0.68dc
0.77bc
1TA8, TB8, and TC8 are TiO2 coatings with binders A, B, and C at 1:8 NP to
binder weight ratios.
Mean values with same low case superscript within the same variable are not
significantly different (P>0.05)
198
Fig 6.1. Effect of type and concentration of binder on the log reduction of E.coli
O157:H7 by TiO2 nanocoatings at 0.5 mW/cm2 UV-A light intensity for 3 h.
Where, UV-A is the plain SS coupon under UV-A light. Binder-A, Binder-B, and Binder-
C are the coatings with binders-A, B, and C under UVA light. TA4 is the TiO2 coatings
with binders A at 1:4 NP to binder weight ratio under UV-A light. TA8, TB8, and TC8
are the TiO2 coatings with binders A, B, and C at 1:8 NP to binder weight ratios under
UV-A light. While, TB16, and TC16 are the TiO2 coatings with binders B, and C at 1:16
NP to binder weight ratios under UV-A light. Error bars represent +/- standard deviation
of 3 measurements. Means not labelled with the same letter are significantly different
(P≤0.05)
G GF F
C
E
E D
A
B
A
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Log r
educt
ion (
CF
U/c
m2)
Treatment type
199
Fig 6.2. Effect of UVA light intensity on the log reduction of E.coli O157:H7 by TiO2
nanocoatings.
Where, UVA is the plain stainless steel under UVA light, Binder-A, Binder-B, and
Binder-C are the coatings with binders-A, B, and C under UVA light. TA8, TB8, and
TC8 are the TiO2 coatings with binders A, B, and C at 1:8 NP to binder weight ratio.
Error bars represent +/- standard deviation of 3 measurements. Means not labelled with
the same letter are significantly different (P≤0.05)
KJL
L
KJI
L
B
GJI
H
BC
A
KL K
JL
KJI
L
CD
GFH
A A
KJI
H
F
GF
A
E
A A
KJI
L
GIH
ED
ED
BC
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
UVA Binder-A Binder-B Binder-C TA8 TB8 TC8
Log r
educt
ion (
CF
U/c
m2)
Treatment type
0.25 mW/cm2 for 180 min 0.5 mW/cm2 for 180 min
0.75 mW/cm2 for 180 min 0.75 mW/cm2 for 90 min
200
Fig 3. Bactericidal activity of different TiO2 nanocoatings against E.coli O157:H7 before
and after repeated use experiment at 0.5 mW/cm2 for 3 h.
Where TA8, TB8, and TC8 are TiO2 coatings with binders A, B, and C at 1:8 NP to
binder weight ratio. The number followed by the sample code is the number of times
coating subjected for repeated use experiment. Error bars represent +/- standard deviation
of 3 measurements. Means not labelled with the same letter are significantly different
(P≤0.05)
D D
A
D
A
C
BC B BC
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
TA8 TA8-1 TB8 TB8-1 TC8 TC8-1 TC8-3 TC8-5 TC8-10
Log r
educt
ion (
CF
U/c
m2)
Treatment type
201
CHAPTER-7
CONCLUSIONS
In this research, physically stable TiO2 nanocoatings with strong photocatalytic
bactericidal property were developed on food contact surface of stainless steel through a
systematic approach. Initially, a testing protocol was developed in suspension to select
bactericidal TiO2 NPs. Type and source of TiO2 NPs, bacterial cell harvesting conditions,
volume of reaction mixture, and the intensity of UVA light were found to have
significant effect on the log reduction. As per this study, a 20 mL of suspension with
single wash of bactericidal cells and 2 mW/cm2 UVA light intensity was found to be the
best testing protocol to evaluate the bactericidal efficacy of TiO2 NPs. In addition, it was
also found that photocatalytic oxidation of organic dyes can be used as a quick and easier
way to screen bactericidal TiO2 NPs prior to actual microbiological tests. Later, the effect
of food processing organic matter on the photocatalytic bactericidal efficacy of TiO2 NPs
was studied using produce and meat wash solutions. Factors such as turbidity, total
phenolics, and protein content of the organic matter were found to have significant effect
on the bactericidal efficacy of TiO2. Also, a linear correlation was observed between
chemical oxygen demand (COD) and total phenolics as well as COD and protein
contents. Further, an empirical equation with COD as predictor variable was proposed to
predict the bactericidal efficacy of TiO2 in the presence of food processing organic
matter. These results would help to take effective strategies to improve the bactericidal
property of TiO2 NPs in food processing environment as a coating.
202
A coating method was developed to create TiO2 nanocoatings on stainless steel using
different binding agents. Shellac, polyurethane, and polycrylic were found to be three
most promising binders to develop physically stable TiO2 nanocoatings on stainless steel
when used at 1:4 to 1:16 NP to binder weight ratios. SEM analysis of the coated surfaces
revealed that by increasing the binder concentration in the coating decreased the amount
of surface exposed TiO2 NPs. An optimum concentration of TiO2 NPs to binder is
required to achieve good physical stability and strong bactericidal property. Overall, TiO2
nanocoating with polycrylic showed highest physical stability followed by TiO2 coating
with polyurethane and shellac when subjected to adhesion, scratch and wear resistance
tests.
An indented coupon technique was found to be the most appropriate to test
photocatalytic bactericidal property of TiO2 nanocoatings. Type of binder used in the
coating has significant effect on the bactericidal property of TiO2 nanocoatings.
Increasing the concentration of binder in the TiO2 coating has decreased the bactericidal
property. A layer-by-layer coating method improved to expose more NPs on the surface
has significantly increased the bactericidal property of TiO2 nanocoatings. However,
further studies are needed to optimize this technique to achieve high durability. After 3 h
photocatalytic disinfection treatment, TiO2 coatings with polycrylic as binding agent
showed highest log reduction followed by TiO2 coating with polyurethane, and shellac,
respectively. Intensity of UVA light has significant effect of the bactericidal property of
TiO2 nanocoatings. However, a light intensity of 0.50 mW/cm2 was found to be the
optimum to exhibit high photocatalytic disinfection efficacy and also to avoid the effect
of UVA light itself on bacterial reduction. On repeated use experiments, TiO2 coating
203
with polycrylic at 1:8 NP to binder weight ratio was found to be physically more stable
with high bactericidal property. The results of this research has showed promise to
develop durable TiO2 nanocoatings with strong bactericidal property on food contact
surfaces using appropriate binding agents.
204
APPENDIX-A
STRATAGIES TO IMPROVE PHOTOCATALYTIC BACTERICIDAL
PROPERTY OF TiO2 NANOCOATINGS
205
1. Objective
To determine the effect of coating method on the bactericidal property of TiO2
nanocoatings.
2. Hypothesis
A modified coating method to expose more nanoparticles (NPs) on the surface of coating
may significantly increase the bactericidal efficacy of TiO2 nanocoatings.
3. Rationale
Studies on TiO2 nanocoatings based on Chapters 5 and 6 under different test
conditions revealed that the type of binder used in the coating has significant effect on the
physical stability and photocatalytic bactericidal property. These differences in the
bactericidal property can be attributed to the differences in surface characteristics of TiO2
nanocoatings created using different binding agents. It is also shown that when TiO2 NPs
were mixed with different binding agents for subsequent coating on stainless steel
surface, majority of the coated surface was covered only with the binder and very less
number of TiO2 NPs were actually exposed on the surface. Since the photocatalytic
disinfection mechanism is a surface active phenomenon, presence of more number of
TiO2 NPs on the surface of coating significantly improves its bactericidal property. A
layer-by-layer (LbL) coating approach to expose more number of TiO2 NPs on the
surface is one possibility to achieve high disinfection efficiency. In addition, LbL coating
method may help to reduce the UVA light intensity levels required for the activation of
TiO2 NPs and the total treatment time required to achieve desired bacterial reduction.
206
4. Methodology
Indented stainless steel coupons as described in Chapter 5 were used for the
coating. In this study, two different approaches were followed for creating TiO2 coating
(Fig. A1). In the first approach, 0.25 ± 0.01 g of TiO2 coating suspension at 1:8 NP to
binder weight ratio which is prepared as described in Chapter 5 was deposited in to the
well of SS indentation by placing the coupon on a calibrated balance. The deposited
suspension was evenly spread across the entire area of the indentation by slowly tilting
the coupon sideways or if needed using Crayola paint brush by maintaining the constant
weight of deposited coating. Later, the coated coupons were air-dried over night at room
temperature. This approach of coating was refereed as direct coating method. In the
second approach, a layer of binder coating equivalent to the weight of binder used in
coating formulation of direct coating method was first dispensed inside the well of
indentation. Immediately, the coupon was placed underneath a sieve (U.S. mesh # 60)
which was fixed with a template of indented coupon on the top and TiO2 NPs (equivalent
weight in coating formulation of direct method) was pushed through the sieve. In this
way a uniform layer of TiO2 NPs were spread on top of the binder layer. After that, the
coupon was removed out and the NPs spread on top of binder layer were slightly pressed
to adhere and strongly attach to the binder coating. Later, the coatings were air-dried over
night at room temperature and tapped off loosely bound NPs on coating before weighing
them on a calibrated balance mentioned before. This approach of coating was referred as
layer-by-layer (LbL) coating method (Fig. A1). E.coli O157:H7 (5-strain) was prepared
at around 107 CFU/mL cell concentration and used in the photocatalytic disinfection
treatment by following methodology reported in Chapter 6.
207
5. Results
Fig. A2 compares the bactericidal efficacy of TiO2 nanocoatings with binders A,
B, and C prepared by the direct coating (TA8, TB8 and TC8) vs the layer-by-layer
coating (AT8, BT8 and CT8) methods. As reported in the Chapter 6, increasing the
intensity of UVA light from 0.25 to 0.75 mW/cm2 significantly increased bactericidal
activity of TiO2 nanocoatings with binder A (TA8) and B (TB8). However, no significant
effect of light intensity has been observed for TiO2 nanocoating with binder C (TC8).
This is attributed to the inherent bactericidal property of binder-C under UV-A light as
discussed earlier in Chapter 6. This shows that the TiO2 nanocoatings created by direct
coating method with binder-C (TC8) have highest bactericidal activity followed by
binder-B (TB8) and binder-A (TA8) (Fig. A2).
By using Layer-by-Layer (LbL) coating approach, TiO2 coating on binder-A
(AT8) showed up to 389, 227, and 137% increase in the log reduction at UVA light
intensities of 0.25, 0.5, and 0.75 mW/cm2, respectively when compared with the direct
coating method (TA8) (Fig. A2). This can be explained by the fact that the TiO2 NPs are
masked in presence of binder while they are freely available for contact with bacteria in
the absence of binder. Faure et al (2011) reported that the bactericidal efficacy of
coatings involving mixture of TiO2, zeoliths, and inorganic binders on a commercial
support material is much less than the TiO2 coatings on quartz support without any binder
even at 87.5% less concentration of TiO2 NPs. No significant difference in the log
reduction of TiO2 nanocoatings on binder-C (CT8) was observed with respect to coating
method after 3 h treatment (Fig. A2). However, method of coating has significant effect
on the log reduction when the treatment time was reduced to only 90 min (CT8>TC8).
208
Whereas, LbL coating method did not improved the bactericidal activity of TiO2 coatings
on binder-B (BT8) when compared with direct coating method (TB8). In contrast, the
bactericidal activity of BT8 decreased by 35 % (at 0.5 mW/cm2) and 5 % (at 0.75
mW/cm2) when compared with TB8. This result can be explained based on our
observation while developing TiO2 nanocoating with binder-B using LbL approach. We
have noticed that the TiO2 NPs sprinkled on the surface of binder-B layer eventually
submerged in the binder layer resulting in fewer surface exposed TiO2 NPs (Fig. A3).
This further reduced the bactericidal property of original TiO2 nanocoatings (TB8)
created by direct coating method even at comparatively higher UVA light intensities.
6. Conclusions
Direct coating of TiO2 by mixing the NPs together with a binding agent as a
suspension may help to achieve high physical stability and durability. However, this
approach of coating limits the photocatalytic bactericidal efficacy of TiO2 nanocoatings
due to possible shielding of binder on the surface of TiO2 NPs. This condition limits the
extent of ROS generated by TiO2 NPs during photo-treatment and takes longer times to
achieve desired disinfection level. On the other hand, increasing the available surface
area of NPs on the surface through Layer-by-Layer coating method significantly
increased the photocatalytic bactericidal property of TiO2 nanocoatings using different
binders. However, this property depends on the nature of the binder used in the coating.
Moreover, binding agents are required to form strong bond with the NPs to achieve
physically stable and durable coatings for use in commercial applications. Further studies
need to be conducted in order to optimize the process of Layer-by-Layer coating to
209
achieve high bactericidal property of TiO2 nanocoatings without affecting the coating
physical stability.
References:
Faure, M., Gerardin, F., André, J.-C., Pons, M.-N., & Zahraa, O. (2011). Study of
photocatalytic damages induced on E. coli by different photocatalytic supports
(various types and TiO2 configurations). Journal of Photochemistry and
Photobiology A: Chemistry, 222(2–3), 323-329.
210
Fig A1. TiO2 nanocoating on stainless steel surface (SS) using (a) Direct coating, (b)
Layer-by-Layer coatings methods
211
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
TA8 AT8 TB8 BT8 TC8 CT8
Log r
educt
ion (
CF
U/c
m2)
Treatment type
0.25 mW/cm2 for 180 min 0.50 mW/cm2 for 180 min
0.75 mW/cm2 for 180 min 0.75 mW/cm2 for 90 min
Fig A2. Effect of coating method on bactericidal activity of TiO2 coatings
Where TA8, TB8, and TC8 are TiO2 coatings with binders A, B, C at 1:8 NP to binder weight
ratios, respectively as deposited by direct coating method; AT8, BT8, and CT8 TiO2 coatings
with binders A, B, C at 1:8 NP to binder weight ratios, respectively as deposited by layer-by-
layer coating method.
212
Fig A3. Comparison of TiO2 nanocoatings with binders A (TA8/AT8), B
(TB8/BT8), and C (TC8/CT8) at 1:8 NP to binder weight ratio created by (i)
Direct coating method (TA8, TB8, and TC8), and (ii) Layer-by-Layer coating
method (AT8, BT8, and CT8).
213
APPENDIX-B
STUDIES ON BACTERICIDAL ACTIVITY OF VISIBLE LIGHT ACTIVATED
IRON OXIDE (Fe2O3) NANOPARTICLES AND NANOCOATINGS
214
1. Objective
To understand the bactericidal properties of iron oxide (Fe2O3) nanoparticles (NPs) in
suspension and as a nanocoating.
2. Hypothesis
Fe2O3 NPs and nanocoatings with potent bactericidal property under visible light
activation can be created using various chemical and physical synthesis and deposition
techniques.
3. Rationale
Fe2O3 has attracted a lot of attention for photocatalytic applications due to its short
band gap energy (Eg = 2.2 eV) and the ability to absorb a large part of the visible light
spectrum (λ= 564 nm). In addition, Fe2O3 NPs are chemically stable, non-toxic, cheap,
and readily available. Very limited information is available in the literature regarding
photocatalytic bactericidal properties of Fe2O3 NPs. Recently, Basnet et al (2013)
reported that the physical vapor deposited (PVD) Fe2O3 nanocoatings has showed
excellent bactericidal property under visible light illumination. They also reported that
the oblique angle deposited (OAD) coatings are more bactericidal compared to thin film
(TF) coatings. However, the effect of method of NP synthesis and deposition techniques
on bactericidal property of Fe2O3 NPs in suspension as well as in coating form is still not
clear. Understanding the effect of fabrication method on bactericidal property of visible
light activated Fe2O3 NPs would be a great use in several food safety applications.
215
4. Methodology
4.1. Fe2O3 NPs and nanocoatings
Several samples of chemically synthesized, ball milled, silicon dioxide (SiO2) and
tungsten oxide (WO3) doped Fe2O3 NPs of known characteristics and photocatalytic
activity were provided by Prof. Yiping Zhao’s lab at the Nanoscale Science and
Engineering Center, The University of Georgia, Athens, GA. In addition, sputter
deposited thin film (TF), OAD nanorod (NR), SiO2 or WO3 doped Fe2O3 coatings were
also provided by Prof. Zhao’s lab. A commercial sample Fe2O3 NPs (Alfa Aesar®, Ward
Hill, MA) was also included for comparison.
4.2. Bacterial strains & inoculum preparation
Bacterial strains either E.coli (ATCC 1428) or E.coli O157:H7 (5-stain) at around
107-8
CFU/mL were used in these studies. Bacterial inoculum for photocatalytic
disinfection treatments was prepared by following the procedure described in Basnet et al
(2013).
4.3. Photocatalytic disinfection in suspension:
Photocatalytic bactericidal activity of Fe2O3 NPs in suspension was determined by
following method described in Yemmireddy and Hung (2015). However, UVA light
source was replaced with a visible light (UTILITECH) in the current study. The
experiments were conducted at different concentration of NPs (1 to 10 mg/mL), volume
of reaction mixture (5 to 30 mL), and light intensities (25 to 100 mW/cm2). One positive
(only aqueous suspension of NPs in dark) and a negative (only visible light without NPs)
control samples were also included in each treatment. The log reductions over a 3 h
216
treatment time were determined by plating appropriate dilutions on tryptic soy agar
(TSA) or sorbital macconkey agar (SMAC) followed by incubation at 37°C for 24 h.
4.4. Photocatalytic disinfection as a coating
Fe2O3 coated glass coupons were first pre-sterilized under 30W germicidal UV
light (254 nm) in a biological safety cabinet (Class II Type A/B3, NuAire, Inc.,
Plymouth, MN) for about 1 h. The sterilized coupons were placed in a 90 mm diameter
petri-dishes containing moistened filter paper at the bottom to prevent drying-out of the
bacterial culture during the treatment. Later, a 100 µL aliquot of bacterial culture was
uniformly spread across the entire surface of Fe2O3 coating using a sterile disposable
loop. These samples were treated under visible light illumination at 50 to 100 mW/cm2
for up to 2- 4 h. The light intensity reaching the surface of each coupon was measured
with a optical power meter (ThorLabs PM100D/S310C). Appropriate positive and
negative controls were also included. During the treatment the filter paper is frequently
moistened to avoid drying-out of inoculum. After the specified treatment time, the
coupons were taken out and immersed in 10 mL of sterile PBS solution containing 0.1%
tween 80 and vortexed for 30 s to re-suspend the bacteria. A viability count (log
CFU/cm2) was performed by appropriate dilution and plating on either TSA or SMAC
and incubation at 37 °C for 24 h.
5. Results
The treatment conditions and the results of various experiments involving Fe2O3
NPs and their doped structures in suspension as well as in coating were summarized in
Table B1. The results indicate that under tested conditions, Fe2O3 NPs in suspension were
not shown significant bacterial reduction. In the first stage, several studies have been
217
conducted using commercial and chemically synthesized Fe2O3 NPs in suspension at
different concentration of NPs, volume of reaction mixture, visible light intensity, initial
bacterial concentration, and photocatalytic treatment time in order to improve the
bactericidal efficacy and to optimize test conditions for maximum bactericidal property.
However, even after subjecting to variable test conditions the tested Fe2O3 NPs were
unable to exhibit significant reduction. At this stage we suspected that the method of
synthesis of NPs may have an effect on their photocatalytic activity. In the second stage
of experiments, Fe2O3 NPs synthesized by ball milling (physical synthesis method) were
again tested for photocatalytic bactericidal property in suspension under different test
conditions. The results did not show an improvement in bactericidal property when
compared with chemically synthesized Fe2O3 NPs. At this stage we suspected that the
charge separation efficiency of Fe2O3 NPs might be severely hindered in the suspension.
As a reason the recombination rate of photo-generated valence band holes and conduction
band electrons were so high that not enough reactive oxygen species (ROS) were
generated from Fe2O3 NPs for bacterial inactivation. Later, in order to increase the charge
pair separation ball milled Fe2O3 NPs mixed with SiO2 and WO3 NPs. However, no
significant difference in the bactericidal activity was observed. Agglomerated NPs in the
suspension was believed to be one possible reason for the limited photocatalytic
bactericidal property of Fe2O3 NPs and their doped powders. Studies reported by Tuchina
et al (2014), and Zhang et al (2011) further support this hypothesis. In the next stage,
inactivation studies were focused on using immobilized Fe2O3 NPs on glass substrate as a
coating. The results indicate that only Fe2O3 NR coatings are showing good bactericidal
activity followed by Fe2O3 thin films further supporting results of Basnet et al (2013).
218
However, doped structures of WO3 core and Fe2O3 shell NRs and WO3-Fe2O3 side coated
NRs were unable to exhibit bactericidal property. Photocatalytic activities by dye
degradation of different nanostructures as provided by Dr. Yiping Zhao’s lab are in the
order of TiO2 NRs/NPs under UVA light >> Fe2O3 NRs under vis-light > Fe2O3 NPs
under vis-light > TiO2 NPs under vis-light. The same trend can be attributed to the high
bactericidal activity of Fe2O3 NRs when compared to Fe2O3 NPs.
6. Conclusions
Fe2O3 is a promising photocatalyst for several food safety applications due to its non-
toxicity and ability to activate under visible light. However, developing Fe2O3
nanostructures on different surfaces using simple approaches for practical application is a
challenge. We made an effort to understand the bactericidal property of Fe2O3 NPs in
suspension and create nanocoating with strong bactericidal activity. We investigated the
effect of fabrication method of Fe2O3 NPs and nanocoatings on bactericidal activity under
different test conditions. Our preliminary results shows that Fe2O3 NPs in suspension are
photo-catalytically not very active to exhibit significant bacterial reduction in contrast to
Fe2O3 nanorod coatings. The possible reason for the differences in photocatalytic
bactericidal activity of Fe2O3 under visible light activation is still not fully understood.
More systematic studies need to be conducted at mechanistic point of view to better
understand the Fe2O3 bactericidal properties under different scenarios.
219
References
Basnet, P., Larsen, G. K., Jadeja, R. P., Hung, Y.-C., & Zhao, Y. (2013). alpha-Fe2O3
Nanocolumns and Nanorods Fabricated by Electron Beam Evaporation for
Visible Light Photocatalytic and Antimicrobial Applications. Acs Applied
Materials & Interfaces, 5(6), 2085-2095.
Tuchina, E. S., Kozina, K. V., Shelest, N. A., Kochubey, V. I., & Tuchin, V. V. (2014).
Iron oxide nanoparticles in different modifications for antimicrobial phototherapy.
Proc. Of SPIE, Vol (8955), 1-12.
Zhang, W., Rittmann, B., & Chen, Y. (2011). Size Effects on Adsorption of Hematite
Nanoparticles on E. coli cells. Environmental Science & Technology, 45(6), 2172-
2178.
220
B1. Summary of photocatalytic bactericidal activity of various types of Fe2O3 in
suspension and as a nanocoating
Type of Fe2O3 NPs Type of
study
Type of bacterial
culture
Test conditions
(Volume of
suspension, light
intensity)
Log reduction/
treatment time
Chemically
synthesized
Suspension E.coli (ATCC 1428) 20 mL
7 mW/cm2
<0.5 log/ 2 h
Commercial &
Chemical
Suspension E.coli (ATCC 1428) 30 mL
65 or 100 mW/cm2
<0.25 log/ 4 h
Ball milled Suspension E.coli (ATCC 1428) 30 mL
100 mW/cm2
<0.25 log/ 3 h
Ball milled Fe2O3
mixed with SiO2
Suspension E.coli (ATCC 1428) 5, 10, 20 mL
100 mW/cm2
<0.25 log/ 2-6 h
Ball milled Fe2O3 -
WO3
Suspension E.coli (ATCC 1428) 10, 20 mL
100 mW/cm2
0.71 log / 3 h
Ball milled WO3-
Fe2O3
Suspension E.coli (ATCC 1428) 10, 20 mL
100 mW/cm2
0.46 log/ 3 h
Commercial
Suspension E.coli O157: H7 20 mL
50 mW/cm2
<0.5 log/ 3 h
Ball milled
Fe2O3- SiO2
Fe2O3 -WO3
Suspension E.coli O157: H7 20 mL
50 mW/cm2
<0.5 log/ 3 h
Fe2O3- WO3 thin film Coating E.coli O157: H7 100 µL
10 mW/cm2
1.15 log / 3 h
Fe2O3 OAD NRs Coating E.coli O157: H7 100 µL
10 mW/cm2
>3.75 log /3 h
WO3 (core) - Fe2O3
(shell) NRs
Coating E.coli O157: H7 100 µL
50 mW/cm2
<0.25 log /2 h
WO3 - Fe2O3 side
coated NRs
Coating E.coli O157: H7 100 µL
50 mW/cm2
<0.25 log /2 h