LWT - Food Science and Technology 61 (2015) 1e6

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Selection of photocatalytic bactericidal titanium dioxide (TiO2)

nanoparticles for food safety applications
Veerachandra K. Yemmireddy, Yen-Con Hung*
Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA

a r t i c l e i n f o a b s t r a c t

Article history: The main objective of this study was to develop a systematic testing protocol for selecting bactericidal
Received 15 September 2014 TiO2 nanoparticles (NPs). Photocatalytic bactericidal activity of TiO2 NPs at 1 mg/mL concentration was
Received in revised form tested against Escherichia coli O157:H7. The effect of source of NPs (three different commercial samples
19 November 2014
referred as T1, T2 and T3), bacterial cell wash conditions (1 vs 3 wash), volume of reaction mixture (10, 20
Accepted 21 November 2014
and 30 mL) and intensity of UVA light (1 vs 2 mW/cm2) on bactericidal activity has been determined.
Available online 28 November 2014
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
Keywords:
TiO2
the overall log reduction (3.27 vs 4.22). Decreasing the volume of reaction mixture increased the log
Nanoparticles reduction. This study has identified the best testing protocol for evaluating TiO2 NP bactericidal efficacy
Photocatalyst as single wash of bacterial cells with a reaction mixture volume of 20 mL and UVA light intensity of
Bactericidal activity 2 mW/cm2. In addition, it was found that photocatalytic oxidation of organic dyes can be used a quick
Escherichia coli O157:H7 and easier way to screen bactericidal TiO2 NPs prior to actual microbiological tests.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction or is restricted by regulations such as pharmaceutical and food

Advanced oxidation processes based on heterogenous photo-
catalysis using photocatalytic nanoparticles (NPs) is gaining
popularity in food safety applications. Heterogenous 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 photo-
catalysts, 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, sta-
ble, 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,
Tsoukleris, Panagou, Falaras, & Nychas, 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
* Corresponding author. Tel.: þ1 770 412 4739; fax: þ1 770 412 4748.
E-mail addresses: kranti@uga.edu (V.K. Yemmireddy), yhung@uga.edu
(Y.-C. Hung).
industries (Skorb et al., 2008). In addition, TiO2 becomes super-
hydrophilic 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 shorter cleaning cycles in the food industry.
Since the photochemical sterilization of Escherichia coli using Pt-
TiO2 was reported by Matsunaga, Tomoda, Nakajima, and Wake
(1985), TiO2 photocatalysts have extensively studied to disinfect a
broad spectrum of microorganisms including viruses, bacteria,
fungi, algae, and cancer cells (Kim, Kim, Cho, & Cho, 2003). The
bactericidal properties of TiO2 are attributed to the high redox
potential of the surface species also known as ROS, such as hydroxyl
radical (
OH), superoxide radical (O$ 2 ), 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, 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,
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 available on the

http://dx.doi.org/10.1016/j.lwt.2014.11.043
0023-6438/© 2014 Elsevier Ltd. All rights reserved.
2 V.K. Yemmireddy, Y.-C. Hung / LWT - Food Science and Technology 61 (2015) 1e6
market and notably, Degussa P25 TiO2, which is considered a
standard and often used as a comparison in scientific experimen-
tation for determining photocatalytic activity (Mills & Le Hunte,
1997). Hoffmann, Martin, Choi, and Bahnemannt (1995) have sug-
gested 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
(Hitkova et al., 2012; Kim et al., 2003). However, none of these
studies have considered important factors like bacterial cell har-
vesting conditions and reaction mixture volume that may nega-
tively 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
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 ob-
jectives include: i) to determine the effect of type and source of TiO2
NPs on bactericidal activity, ii) to determine the effect of cell har-
vesting conditions, light intensity and volume of suspension on
bactericidal activity.
2. Materials and methods
2.1. Nanoparticles
Total three different types of commercial TiO2 NPs (referred as
T1, T2 and T3) of known characteristics (Table 1) were used in this
study. Samples T1 and T2 were of anatase crystal phase with
10e25 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 were listed in Table 1. Aqueous sus-
pensions 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 photo-
catalytic 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 shaking incubator at
Table 1
Characteristics of commercial TiO2 NPs.
TiO2 samplea Crystal phase Purity (%) Size (nm) Specific surface True
area (m2/g)
T1 Anatase 99.5 10e25 50e150
T2 Anatase >99 10e25 200e240
T3 Anatase-Rutile 99.5 ~21 35e65
a
Commercial TiO2 samples T1: Sky Spring Nanomaterial's; T2: US Research
Nanomaterials; T3: Degussa P-25 from Aldrich.
230 rpm and 37  C for 16 h. Following the incubation, cells were
harvested by sedimentation either once (4000  g for 12 min) or
resuspended and sedimented three times (3200  g for 10 min) in
sterile phosphate-buffered saline (PBS, pH 7.2) in order to deter-
mine the effect of cell harvesting conditions on the photocatalytic
disinfection efficacy. The harvested cells were re-suspended in
10 mL PBS and equal volume 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
UV/Vis spectrophotometer (Beckman DU520, Beckman Coulter Inc.,
Brea, CA, USA) and confirmed by plating 100 mL portions of the
appropriate serial dilution on tryptic soy agar (TSA) (Difco Labo-
ratories) 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  18 mm2; diameter  depth) mounted
on a magnetic stirrer (Model# H1190M, Hanna Instruments,
Smithfield, RI, USA) together placed in a photocatalytic disinfection
chamber (Fig. 1). Aqueous suspension of TiO2 NPs of 9, 18 and 27 mL
volume 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 indi-
vidually. 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
HP™, LL-UV P40, Los Angeles, CA, USA) from the top under
continuous stirring at medium speed with a magnetic stirbar
(3.8 cm length  1.25 cm diameter) (Fig. 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 photo-
catalyst under UVA light) control samples were also included. All
the experiments were conducted at room temperature using in-
door air as oxidant. A sample of 1 mL was withdrawn from the
density
(g/cm3)
NA
3.9
NA
Fig. 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.
 V.K. Yemmireddy, Y.-C. Hung / LWT - Food Science and Technology 61 (2015) 1e6
treatment solution at every 30 min for 3 h and added into 9 mL
sterile PBS. Appropriate serial dilutions of the samples were pre-
pared and the surviving bacteria from the control and treatments
were enumerated by spiral plating 50 mL 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 evalu-
ated by degradation of methylene blue (MB) solution. The photo-
decay rate was measured by using 20 mL of TiO2 aqueous solution
(1 mg/mL) saturated with MB dye (20 mg/L) in a petridish illumi-
nated with UVA light at 2 mW/cm2 under continuous stirring as
described earlier. A 3 mL of the 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 UVeVis spectrophotometer. Residual concentration
of MB (mg/L) due to TiO2 photocatalytic activity was calculated by
using standard MB curve.
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 Soft-
ware Release 9.3 (SAS Institute). 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 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 was shown in Fig. 2. Under tested conditions, Sample T3
has exhibited highest log reduction (5.78) followed by T2 (2.59) and
T1 (1.81), respectively. This indicates that the type and source of
TiO2 has played an important role on the log reduction. In
Fig. 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.
3
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 1). Sample T1 and
T2 used in this study have a wide particle size distribution of
10e25 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. (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 current study,
though sample T1 and T2 have similar size distribution
(10e25 nm), T2 has 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
(200e240 m2/g) compared to T1 (50e150 m2/g). Increasing the
particle surface area provides high relative OH ion coverages for
hydroxyl radical (OH$) formation which is an important ROS
responsible for photocatalytic disinfection. In contrast, sample T3
with mixed phase of anatase (~80%) and rutile (~20%), an average
particle size of ~21 nm and relatively low SSA (35e65 m2/g) have
showed highest bactericidal activity than T1 and T2.
As reported in several studies, high bactericidal activity of mixed
phase of anatase-rutile TiO2 powder, particularly Degussa P-25
(sample T3) attributed to the generation of high amount of ROS
such as OH$, O-2 and H2O2 due to effective charge separation by
avoiding electronehole 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 inacti-
vation. Another study reported that Degussa P-25 obtained by
flame pyrolysis (Degussa, 1997) is an effective photocatalyst for
bacterial inactivation. Bickley, Gonzalez-Carreno, Lees, Palmisano,
and Tilley (1991) reported that the dynamic process of Degussa P-
25 preparation would create a complex variety of multiphased
particles characterized by juxtaposition of anatase with rutile
phases. This condition results in increased charge separation and
slows down electron (e) e hole (hþ) pair recombination rate
which in-turn results in high photocatalytic activity. Nguyen, Amal,
and Beydoun (2005) reported for the same mass of NPs, dispersed
particle sizes of a commercial TiO2 PC 500 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 prepara-
tion method of the TiO2, NP surface structure, size, and crystallo-
graphic structure all played an important role 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 and source of TiO2 on bactericidal activity.
Degussa P-25 was found to be the 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 photo-
catalytic 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
4 V.K. Yemmireddy, Y.-C. Hung / LWT - Food Science and Technology 61 (2015) 1e6
reaction mixture volume was shown in Fig. 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 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 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 in 3 h respec-
tively 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, Sharma, van der Mei, and Busscher (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 exper-
iments. Gilbert, Evans, Evans, Duguid, and Brown (1991) reported a
decrease of 25 and 40% in the viability of exponential-phase Pseu-
domonas aeruginosa following centrifugation at 5000  g and
10,000  g respectively. Similar experiments with stationary and
exponential phase E. coli cells greatly altered biocide sensitivity
(Gilbert, Pemberton, & Wilkinson, 1990). Pembrey, Marshall, and
Schneider (1999) reported loss of viability and modification of
physicochemical cell surface properties of E. coli or Staphylococcus
epidermis by high-speed centrifugation (15,000  g) when
compared to harvesting at (5000  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 treat-
ment. Especially, increasing the number of cell washes resulted
higher bactericidal efficacies by less effective commercial samples
T1 and T2 (Fig. 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 bacteri-
cidal 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 wavelength) intensity on the
log reduction of TiO2 samples (T1, T2 and T3) was shown in Table 2.
Increasing the light intensity from 1 mW/cm2 to 2 mW/cm2
increased overall log reduction of TiO2 NPs from 3.27 to 4.22.
Although the trend of bactericidal efficacy is unchanged (i.e.
T3 > T2 > T1), the intensity of UVA light has showed significant
effect on efficacy of individual TiO2 samples. Increasing the UVA
intensity from 1 to 2 mW/cm2 significantly increased the log
reduction of T1 (2.04e3.04), T2 (2.99e3.94) and T3 (4.77e5.67).
Benabbou, Derriche, Delix, Lejeune, and Guillard (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 intensity increases the amount of photon
generation which results in more electronehole pairs formation,
eventually leading to the formation of more OH radicals (Marugan,
van Grieken, Cassano, & Alfano, 2010). On the other hand, Cho,
Chung, Choi, and Yoon (2004) reported the existence of linear
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 (Herrmann,
1999). However, it is believed that the tested intensity range
(1e2 mW/cm2) of current study operating in the linear region
corresponding to the optimal light utilization. This implies that the
saturation of TiO2 acting as the photosensitizer has not been
reached when increasing intensity from 1 to 2 mW/cm2. Benabbou
et al. (2007) noticed an induction period in the first 10 min for the
lower intensities, which suggests that the self-defense and auto-
repair mechanism for protecting the bacteria were more efficient
at a low intensity, but still insufficient as regard the accumulation of
irradiation. Similar results were also observed in the current study
with an initial lag period of ~30 min at low intensity (i.e. 1 mW/
cm2) for the photocatalytic disinfection (data not shown). To
further evaluate the photocatalytic disinfection efficacy of TiO2 NPs,
the effect of volume of suspension has also been investigated for
optimal light utilization.
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 2. Statistical analysis of the data revealed that the overall log
reduction of TiO2 is higher (4.32) at lower volume (10 mL) when
compared to higher volumes (20 and 30 mL). No significant dif-
ference in the reduction has been observed between 20 and 30 mL
volume of suspensions at the end of 3 h treatment time. As ex-
pected, all the tested TiO2 samples (T1, T2 and T3) exhibited high
reduction 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). Whereas,
sample T2 has showed significantly higher reduction (4.30) at
10 mL and almost similar reductions at 20 and 30 mL (~3 log). The
most effective sample T3 has only showed significant difference in
the reduction between 10 mL (5.64 log) and 30 mL (4.70 log).
Table 2
Effect of light intensity and volume on bactericidal activity of TiO2 NPs.
Variable Log reductiona (CFU/mL) after 3 h treatment
Overall T1 T2 T3
Intensity (mW/cm ) 2
1 3.27B 2.04B 2.99B 4.77B
2 4.22A 3.04A 3.94A 5.67A
Volume (mL)
10 4.32A 3.02A 4.30A 5.64A
20 3.55B 2.32A 3.09B 5.32AB
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).
 V.K. Yemmireddy, Y.-C. Hung / LWT - Food Science and Technology 61 (2015) 1e6
These results indicate that volume has significant effect on the
log reduction of individual TiO2 NPs. We expected improved effi-
cacy of 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 has been 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 will not improve
photocatalytic disinfection efficacy. To support this hypothesis,
Gumy et al. (2006) reported that out of several surface properties,
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 effective surface area of NP available for bacteria to come
in-contact while stirring the suspension during photocatalytic
disinfection. Further we observed, a minimum of 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 was shown Fig. 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 oxida-
tion. Similar photocatalytic disinfection trend of TiO2 samples (T1,
T2 and T3) was observed for E. coli inactivation (Fig. 3). This shows
that the photocatalytic degradation potential of organic com-
pounds such as MB can be used as a prior screening test to indi-
rectly predict bactericidal efficacy of TiO2 NPs since the mechanism
of both the processes depend on ROS generation rate. Chen, Yang,
Xu, Wu, and Zhang (2009) reported apparent correlation between
the photocatalytic processes of decomposing formaldehyde and
inactivating E. coli. They noticed 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 analogy is potential method to evaluate the
antimicrobial effect based on organic compound degradation effect.
Similarly, Marugan et al. (2010) reported that analogies between
photocatalytic degradation of chemicals and microorganism inac-
tivation were agreeable only when analyzing the effect of
Fig. 3. Comparison of photocatalytic degradation rate of methylene blue and photo-
catalytic disinfection rate of E. coli O157:H7 among different TiO2 NPs. T1: Sky Spring
Nanomaterial's; T2: US Research Nanomaterials; T3: Degussa P-25.
5
operational variables such as catalyst concentration or light in-
tensity. However, different microbiological aspects (osmotic stress,
repairing mechanism, regrowth, bacterial adhesion to TiO2 surface,
etc) makes disinfection kinetics significantly much complex than
the oxidation of chemical compounds (Marugan et al., 2010). Hence
certain analogies 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 applica-
tions prior to actual microbiological tests.
4. Conclusions
Photocatalytic disinfection efficacy of three different commer-
cial TiO2 NPs to inactivate E. coli O157:H7 has been systematically
investigated. Type and source of TiO2 has showed significant effect
on 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 wash prior to photo-
catalytic disinfection treatment increased the log reduction of even
least effective TiO2 samples. It is preferred to use less severe cell
harvest conditions for accurate determination of bactericidal effi-
cacy of TiO2 NPs. As expected, increasing the light intensity
increased the overall log reduction of all TiO2 samples. Volume of
suspension has showed variable effect on efficacy of tested TiO2
NPs. As per 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.
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 Pro-
cessing Technologies to Destroy Foodborne Pathogens Program-
(A4131).
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