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Inactivation of bacteria and yeast using high-
frequency ultrasound treatment
Shengpu Gao a,b,c, Yacine Hemar a,*, Muthupandian Ashokkumar d,Sara Paturel a,b, Gillian D. Lewis b
a School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland, New ZealandbSchool of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealandc Institute of Food and Agricultural Standardization, China National Institute of Standardization, Beijing 10088,
ChinadSchool of Chemistry, University of Melbourne, VIC 3010, Australia
a r t i c l e i n f o
Article history:
Received 30 September 2013
Received in revised form
11 April 2014
Accepted 19 April 2014
Available online 1 May 2014
Keywords:
High-frequency ultrasound
Bacteria inactivation
Yeast
Enterobacter aerogenes
Bacillus subtilis
Staphylococcus epidermidis
Aureobasidium pullulans
a b s t r a c t
High-frequency (850 kHz) ultrasound was used to inactivate bacteria and yeast at different
growth phases under controlled temperature conditions. Three species of bacteria,Enter-
obacter aerogenes, Bacillus subtilis and Staphylococcus epidermidis as well as a yeast, Aur-
eobasidium pullulans were considered. The study shows that high-frequency ultrasound is
highly efficient in inactivating the bacteria in both their exponential and stationary growth
phases, and inactivation rates of more than 99% were achieved. TEM observation suggests
that the mechanism of bacteria inactivation is mainly due to acoustic cavitation generated
free radicals and H2O2. The rod-shaped bacteriumB. subtiliswas also found to be sensitive
to the mechanical effects of acoustic cavitation. The study showed that the inactivation
process continued even after ultrasonic processing cessed due to the presence of H2O2,
generated during acoustic cavitation. Compared to bacteria, the yeast A. pullulans was
found to be more resistant to high-frequency ultrasound treatment.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Water is extensively used in the food industry, from being an
ingredient, transport medium to a sanitation tool. Water
quality and food safety are inextricably linked and serious
diseases and illness to human beings will be caused if water is
contaminated by bacteria and other microorganisms which
cause diseases. Chlorine disinfection is widely used to
eliminate microorganisms in water through chemical oxida-
tion (Cheremisinoff and Cheremisinoff, 1993). However, some
bacteria are chlorine-tolerant, and need higher chlorine level
or prolonged chlorination time during water treatment. This
results in the development of unpleasant flavours due to the
formation of chlorophenols (Drakopoulou et al., 2009; Phull
et al., 1997). In some cases, certain bacteria are hardly inacti-
vated by chlorination as they form clusters (Phull et al., 1997).
As a result, the use of ultrasound treatment can be considered
* Corresponding author. Tel.: 64 9 9239676; fax: 64 9 3737422.
E-mail address: [email protected](Y. Hemar).
Available online atwww.sciencedirect.com
ScienceDirect
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as a potential method for the inactivation of microorganisms
in aqueous solutions, liquid foods and for the treatment of
wastewater.
The use of ultrasound in bacteria inactivation was first re-
ported in the 1920s (Harvey and Loomis, 1929), and the
fundamental mechanism of bacterial inactivation was
explored during the 1950s and 1960s (Davies, 1959; Earnshaw
et al., 1995). Biocidal effects of ultrasound treatment are sug-gested to be due to mechanical effects and sonochemical re-
actions produced by acoustic cavitation (Ashokkumar, 2011;
Butz and Tauscher, 2002; Earnshaw et al., 1995; Mason, 1990;
Mason et al., 2005; Russell, 2002; Sala et al., 1995; Wu and
Nyborg, 2008). Acoustic cavitation is the formation, growth
and collapse of microbubbles in liquid media. When ultra-
sound passes through liquid media, cavitation bubbles are
generated, which collapse when they reach their critical size
(Leighton, 1994). The mechanical effects such as shear forces
and micro-jettings are produced during the collapse of cav-
ities, andthe energy released can damage bacteria (Colliset al.,
2010; Joyce et al., 2003a; Mason et al., 2003, 2005; ODonnell
et al., 2010; Soria and Villamiel, 2010; Wu and Nyborg, 2008).Although cavitation can be produced during both low-
frequency and high-frequency ultrasound treatments, the
energy released during the collapse of cavities is different at
these frequencies. Low-frequency ultrasound generates larger
cavitation bubbles and their collapse results in a higher energy
release compared to high-frequency ultrasound. Note that for
ultrasound treatment, high-frequency is defined as a fre-
quency higher than 100 kHz [2, 9e11]. For high-frequency ul-
trasound, in addition to physical forces, more free radicals are
generated compared to low-frequency ultrasound, and this is
attributed to the fact that there are more cavitation events
occurring (Crum, 1995). Energy released at the moment of
cavitation bubble collapse is sufficient to break chemicalbonds, and if the medium is water, then the water molecules
are broken into hydrogen atoms and hydroxyl radials (OH)
(Leighton, 1994). Particularly, hydroxyl radials are markedly
produced during high-frequency ultrasonication (He et al.,
2006). These hydroxyl radials can attack the cell wall mem-
brane, and the recombination of these radicals into hydrogen
peroxide, a well-known bactericide, also contributes to bacte-
ria inactivation (Al Bsoul et al., 2010; Joyce et al., 2011).
Although the inactivation of bacteria by ultrasonic treatment
has been widely examined recently, most of the researches
focus on low-frequency ultrasonic inactivation. In addition,
most studies reported in the literature focused only on one or
two bacteria. The main aim of this study is to determine theeffects of high-frequency (850 kHz) ultrasound treatment on
bacteria and yeast in their different growth phases, and to
obtain a deep insight into the mechanism of microbial inacti-
vation by high-frequency ultrasound treatment.
Microorganisms having different size, shape, and the type
of cell wall (Gram nature) were selected, namely Enterobacter
aerogenes, Bacillus subtilis and Staphylococcus epidermidis and a
yeast Aureobasidium pullulans. E. aerogenes exits widely in
water, dairy products, soil, as well as human and animal
faeces (Grimont and Grimont, 2006).B. subtilis was chosen as
one of the indicators of water treatment efficiency in a water
treatment plant (Huertas et al., 2003). As a common skin or-
ganism (Schleifer, 2009), S. epidermidis is known to easily
adhere to surfaces to form biofilms (Katsikogianni et al., 2006),
which on water pipe surfaces can cause potential microbio-
logical contamination (Wingender and Flemming, 2004). A.
pullulans is found ubiquitously in various environments
including water, soil, wood, rocks with high humidity and
plant materials (Chi et al., 2009; Urziet al., 1999). E. aerogenes is
a gram-negative rod-shaped bacterium from the family of
Enterobacteriaceae and the size range of these bacteria are0.3e1.0 mm 1.0e6.0 mm(Imhoff, 2005). B. subtilis is a gram-
positive rod-shaped bacterium which has a size range of
0.7e0.8 mm 2.0e3.0 mm(Schleifer, 2009). S. epidermidis is a
gram-positive coccus and its diameter is 0.8e1.0 mm (Schleifer,
2009).A. pullulansis a yeast-like fungus, also popularly known
as black yeast (Cooke, 1959; Hoog, 1993), is 2e13 mm wide
(Zalar et al., 2008). Low-frequency ultrasound has been stud-
ied widely for the disinfection of wastewater, involving mi-
crobes such as Escherichia coli XL-1 Blue (24 kHz) (Antoniadis
et al., 2007), E. coli K12 (24e80 kHz) (Paleologou et al., 2007),
total coliforms, faecal coliforms, Pseudomonas spp. faecal
streptococci and Clostridium perfringens species (24 kHz)
(Drakopoulou et al., 2009). The bacteria and yeast consideredin this study were previously investigated for their behaviour
under low-frequency ultrasound treatment (Gao et al.,
2014a,b; Joyce et al., 2003b; Singer et al., 1999). B. subtilis
spores were disrupted by high-frequency ultrasonication
using dual transducers (Warner et al., 2009). The synergic ef-
fect of high-frequency ultrasound and vancomycin on S. epi-
dermidis biofilm was also reported in a study which focused
mainly on the mechanical effects induced by ultrasound such
as the enhancing transportation of vancomycin (Dong et al.,
2013). However, to the best of our knowledge, there are no
reports about high-frequency ultrasonication of E. aeroenes
andA. pullulans.
2. Material and methods
2.1. Materials
Hydrogen peroxide (30%) and t-butanol were obtained from
AR ECP Ltd, New Zealand. Glucono-d-lactone (GDL) was pur-
chased from Sigma Aldrich (USA). GDL was used to adjust the
acidic pH of the bacteria cultures, and t-butanol was used as a
hydroxyl radical scavenger. All other chemicals were of
analytical grade and were purchased from Sigma Aldrich.
2.2. Preparation of microbial suspensions
E. aerogenes,B. subtilis,S. epidermidis, andA. pullulans were ob-
tained from the Microbiology Lab of the School of Biological
Sciences at the University of Auckland, New Zealand. 1 ml
bacterial stock stored at 80 C was incubated overnight on
Nutrient Agar at 37 C by spread method. Then a loop of the
bacterial colony was inoculated into 50 ml Nutrient Broth to be
incubated at 37 C overnight while shaking (200 rev min1).
After incubation, a working bacterial culture was prepared. 4 ml
working culture was thentransferred into100 ml freshNutrient
Broth to be incubated under the same conditions. The expo-
nential phase of each microorganism was measured using a
spectrophotometer as previously described (Gao et al., 2014a).
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The bacteria suspensions at exponential phase were obtained
after 2 h,5 h and 3 h incubationfor E. aerogenes, B. subtilis and S.
epidermidis, respectively, and the bacteria suspensions at sta-
tionary phase were obtained after 20 h incubation. A. pullulans
was incubated at 28 C, and the yeast suspension to be ultra-
sonicated was obtained after 72 h growth.
Physiological salt solution (0.9% NaCl, PSS) was used to
wash microbial suspensions in order to get rid of excess nu-trients while keeping the bacteria and yeast alive. Bacterial
suspensions were centrifuged at 10,000 g for 10 min at 4 C
using a Biofuge Stratos centrifuge (Heraeus, Thermo Electron
Corporation, Germany). The supernatant was discarded and
the pellet was resuspended in PSS. A. pullulans was centri-
fugedat 3000 g for10 min at4 C, and the pellet of each sample
was washed twice. After washing, the initial number of each
species was estimated by measuring the optical density at
600 nm using a HelIOS b UVevisible spectrophotometer
(Thermo Electron Corporation, UK).
2.3. Ultrasound treatment
Bacterial and yeast suspensions were ultrasonicated by an
ultrasound generator K80 (Meinhardt Ultraschalltechnik,
Germany) at 850 kHz, which was connected to an ultrasonic
transducer E/805/T and a double-walled cylindrical glass
vessel (Fig. 1). The vessel was connected to a water bath
(PolyScience SD07R-20-A12E, USA). The glass vessel was filled
with 250 ml Milli-Q water. 5 ml microbial suspensions were
transferred into a 15 ml glass tube. The tube was then inserted
through a PVC cover of the vessel and was positioned in the
centre of the vessel. The lids of the tubes were held tightly by
the PVC cover in order to keep the tubes in the same position.
Circulating water bath (2 C) was used to control the temper-
ature during ultrasonication. Under the ultrasound conditionsused in this study, the working temperature in the vessel
never exceeds 20 C. All ultrasound treatments were per-
formed at least in duplicate.
The ultrasound powerP was determined using the calori-
metric method (Kikuchi and Uchida, 2011; Koda et al., 2003):
P mCp
DT
Dt
(1)
where Cp (4.18 J/(g K)) is the specific heat capacity of water, m
is the mass of ultrasonicated water, DTis the increase in the
temperature, and Dtis the applied ultrasound time.
2.4. Microbial enumeration
A slightly modified MileseMisra method (Miles et al., 1938)
was used to count the viable microbial cells. Unless specified,
bacteria and yeast counts were performed2 h after ultrasound
treatment. For each sample, serial 1:10 dilutions from 100 to
106 times depending on the initial number of microorgan-
isms were made in a 96 Well Tissue Culture plate (Cellstar,
Greiner bio-one, Germany) by mixing PSS with bacterial or
yeast samples. Nutrient Agar plates were marked into six
sections in advance. Each diluted sample (50 ml 3) was
dropped onto three sectors. After that, the plates were incu-bated at 37 C overnight for the bacteria and at 28 C for
24e36 h for the yeast. The countable number (15e150) of col-
onies for each section was counted under a light microscope.
The original bacterial number was calculated using:
N n di 20 (2)
where N is the number of total colonies (CFU/ml), n is the
average number of colonies for a dilution and di is dilution
factor. Note that a large volumeof sample(100 ml 3) was used
when the number of bacteria was low.
2.5. Hydrogen peroxide measurement
A colorimetric method (Alegria et al., 1989; Awtrey and
Connick, 1951; Weissler, 1959) was used for the measure-
ment of the yield of H2O2 by using a UVeVis absorption
Fig. 1e Illustration of the high-frequency unit used in this study. (1) Ultrasound generator, (2) ultrasound transducer, (3)
double-walled cylindrical glass vessel connected, through (4) to the water bath, and (5) test-tube containing the sample.
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spectrophotometer (UV mini 1240, Shimadzu, Japan) at
353 nm wavelength. Potassium idodide (KI) used in this
method was obtained from ECP Ltd, New Zealand, and all
other chemicals were purchased from SigmaeAldrich, USA.
The iodide reagent was made by mixing 1 ml of Reagent A
(3.32 g potassium iodide, 0.10 g sodium hydroxide, 0.01 g
ammonium molybdate tetrahydrate in 50 ml of water) and
1 ml of Reagent B (1.021 g potassium hydrogen phthalate).1 ml of ultrasonicated sample was transferred into 2 ml io-
dide reagents before measuring the absorbance. The con-
centration of H2O2 (c, M) can be calculated by the following
equation:
c A
l 3 (3)
where A is absorbance value at 353 nm, is the molar
extinction coefficient (26,400 M1 cm1), and l is the path
length of cuvette (1 cm).
2.6. TEM
Bacteria samples were stained by 0.2% or 2% aqueous uranyl
acetate before performing examination by Transmission
Electron Microscopy (TEM) (CM12 TEM, Philips, Netherlands).
In order to maintain the surface of carbon-coated copper TEM
grids hydrophilic, the grids were glow discharged at 500 V for
15 s. The discharged grids were immersed into bacteria sus-
pensions for 30 s then the grids were washed quickly twice by
dipping them into Milli-Q water and dried by filter paper. The
dried grids with E. aerogenes and B. subtilis then were
immersed into a drop of 2% uranyl acetate for 30 s, and the
grid withS. epidermidiswas stained by 0.2% uranyl acetate for
10 s. After that the stained grids were dried by fresh filter
papers before microscopy observation.
3. Results and discussion
3.1. Generation of OH and H radicals and H2O2
As mentioned previously, acoustic cavitation induces me-
chanical effects and sonochemical reactions such as the for-
mation of hydroxyl radicals during high-frequency
ultrasonication. For low-frequency high-power ultrasound
treatment of bacteria, inactivation was mainly due to me-
chanical effects induced by acoustic cavitation (Gao et al.,
2014a,b). However in the case of high-frequency ultra-sonication, sonochemical effects (Koda et al., 2009) and the
combination of both mechanical and sonochemical effects
(Hua and Thompson, 2000) were suggested as the main
mechanism for the inactivation of bacteria. Thus, it is
important to obtain an indication about the amount of hy-
droxyl radicals generated by the ultrasound unit. This can be
indirectly quantified by measuring H2O2 yield (Alegria et al.,
1989; Weissler, 1959). The primary sonochemical reactions in
aqueous solutions involve the generation of OH and H radi-
cals and H2O2 (Reactions (4) and (5)) (Dai et al., 2006; Olvera
et al., 2008; Thangavadivel et al., 2009):
H2O ))))/ OH H (4)
2OH/ H2O2 (5)
The iodide method is commonly used to quantify the
concentration of hydrogen peroxide produced during ultra-
sonication (Alegria et al., 1989; Weissler, 1959). Iodide ions are
oxidized by hydrogen peroxide to form molecular iodine
(Reaction 6). The molecular iodine can react with the excess
iodide ions to generate I3 complex (Reaction 7), which can bedetected by UV measurements at 353 nm.
H2O2 2I
/ I2 2OH (6)
I2 I/I3 (7)
Fig. 2 reports the amounts of H2O2 generated in water
during ultrasonication at different ultrasound power at a
constant sonication time of 20 min. A marked increase in the
amount of generated H2O2can be clearly seen for ultrasound
power higher than 10 W. These results are in agreement with
previous studies which showed that high-frequency ultra-
sonication results in an increase of H2O2 with the ultra-
sonication power (Kanthale et al., 2008). It should be notedthat post ultrasonication, the amount of the H2O2 slowly
decreased with time (results not shown). The increase in H2O2with an increase in acoustic power is also mirrored by a
decrease in the pH of the ultrasonicated PSS aqueous solution,
which decreases from a value of 6.0 before sonication to a
value of 3.7 after sonication at 62 W for 20 min (Fig. 2). The pH
was measured because acidic conditions are known to
contribute to the inactivation of some microorganisms such
as E. coli (Salleh-Mack and Roberts, 2007). The pH decrease
during ultrasonication can be explained by the formation of
nitrous and nitric acid (Mead et al., 1976; Supeno and Kruus,
2000), and the formation of carbonic acid by the dissolution
of carbon dioxide by ultrasonication (Semenov et al., 2011).
3.2. Effect of sampling time on bacteria counts
Since the production of OH and H radicals and H2O2 are
known to inactivate microorganisms and that the amount of
the H2O2change with time post ultrasonication as indicated
Fig. 2 e Hydrogen peroxide (-) produced by high-
frequency sonication of Milli-Q water and pH (C) of PSS, as
a function of the ultrasound power. Sonication time was
20 min. Error bars correspond to standard deviations.
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above, it is important to study the effect of sampling time on
the bacteria counts. Sampling time here corresponds to the
time after ultrasonication, at which the ultrasound treated
bacteria cultures were cultured on agar gels in order to
determine the survival rate. A preliminary experiment was
performed where E. aerogenes was ultrasonicated under
different ultrasound power and sonication time conditions.
These conditions corresponded to ultrasonication at 50 W for20 min, 62 W for 10 min and at 62 W for 20 min. A non-
ultrasonicated E. aerogenes suspension was also used as a
control. The ultrasound treatedE. aerogenessuspensions were
kept on ice after ultrasonication and aliquots were drawn at
regular time intervals to be grown on agar plates for viable
bacteria numbering.
Fig. 3 shows the effect of sampling time on the Log(N/N0) of
survival ratio of the non-sonicated and sonicatedE. aerogenes
dispersions. In the case of non-sonicated dispersion there is
no effect of sampling time for up to 12 h (only the first 4 h are
shown in Fig. 3). This is expected since the non-sonicated
suspensions were kept on ice and that their viable number
is not expected to increase nor decrease during that time.However, in the case of bacteria suspensions treated at the
highest acoustic powers and longest time (62 W for 20 min),
there was clearly an effect of sampling time, when compared
to bacteria suspensions sonicated at 50 W for 20 min or 62 W
for 10 min. Under sonication condition of 62 W for 20 min, the
Log(N/N0) of the sonicated dispersions decreased linearly with
an increase in the sampling time. The decrease in viable
bacteria wasalso noticeable forsuspensions sonicated at 62 W
for 20 min, where the decrease in Log (N/N0) was two folds
higher in magnitude when the suspension was left for 200 min
after sonication. This is also expected since the longer the
bacteria suspensions remain in an acidic environment in the
presence of H2O2, the higher is the number of bacteria
inactivated. This result is very important as it demonstrates
clearlythat the sampling time has a direct consequence on the
counting of the number of the viable bacteria. For this reason,
it was decided that in the present work all bacteria counts
would be performed at a constant time (2 h) after sonication,
to ensure reproducibility of the bacteria counting results.
3.3. Ultrasound treatment of bacteria
The effectsof high-frequency (850kHz) ultrasound treatments
of different bacteria at different growth phases were investi-
gated. The initial numbers of the bacteria were approximately
w108 CFU/ml. Fig. 4 shows the survival ratio Log (N/N0) of
E. aerogenes, B. subtilis and S. epidermidis at both exponential
0 50 100 150 200 250 300
-8
-7
-6
-5
-4
-3
-2
-1
0
Sampling time (min)
Log(N/N0)
Fig. 3e Log of survival ratio (Log (N/N0)) ofEnterobacter
aerogenesas a function of storage time (different sampling
time after ultrasonication) for bacterial suspensions
sonicated under different conditions. Control (without
ultrasound treatment) (-); ultrasonicated at 50 W for
20 min (C); ultrasonicated at 62 W for 10 min (:); and
ultrasonicated at 62 W for 20 min (;). Error bars
correspond to standard deviations.
0 10 20 30 40 50 60 70
-5
-4
-3
-2
-1
0
0 10 20 30 40 50 60 70
-5
-4
-3
-2
-1
0
0 10 20 30 40 50 60 70
-5
-4
-3
-2
-1
0 (C)
(B)
Log(N/
N0)
(A)
Log(N
/N0)
Ultrasound Power (W)
Log(N
/N0)
Fig. 4 e Log of survival ratio (Log (N/N0)) for ultrasonicated
bacteria for 20 min as a function of different ultrasound
powers. (A)Enterobacter aerogenes, (B)Bacillus subtilis and
(C)Staphylococcus epidermidis. Symbols are: bacteria at
stationary phase (-); and bacteria at exponential phase
(C). Error bars correspond to standard deviations.
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and stationary phase as a function of ultrasound power. All
results indicated that the bacteria inactivation had been
enhanced with the increasing ultrasound power at both
exponential and stationary phase. There were w4.2, w2.5 and
w4.4 log reductions achieved at 62 W for 20 min ultra-
sonication for E. aerogenes, B. subtilis and S. epidermidis,
respectively, in the stationary phase. Moreover, there was no
marked difference for the inactivation ratio between thebacteria at exponential phase and at stationary phase. In
addition, there was no obvious effect on the bacterial viability
at lowerpower ultrasound treatment (at 9 W or15 W), which is
likely due to the lower concentration of hydroxyl radicals and
hydrogen peroxide produced at lower powers as shown
inFig. 2.
It is likely that the inactivation of the bacteria is due to the
generated hydroxyl radicals and H2O2directly attacking bac-
terial cells. The formation of free radicals during ultra-
sonication was demonstrated previously by electron spin
resonance spectroscopy and spin trapping techniques (Riesz
et al., 1985). It is also known that the effects of free radicals
on cells included damage to DNA, enzymes, liposomes and
membranes (Riesz and Kondo, 1992). For instance, hydroxylradicals are known to oxidize macromolecules presented in
cells, including DNA, lipids and proteins (Cabiscol et al., 2010;
Liu et al., 2011). It is also known that the chemical structure of
bacteria cell wall can be attacked, weakened and then dis-
integrated by the free radicals produced during cavitation
(Joyce et al., 2003b). For example, OH radicals were reported to
Fig. 5 e TEM micrographs of (A) Enterobacter aerogenes, (C)Bacillus subtilis and (D)Staphylococcus epidermidisbefore (1) and
after (2, 3 and 4) ultrasound treatments (20 min 62 W). (B)Enterobacter aerogenes, before (1) and after (2, 3 and 4) the addition
of H2O2(200 mM).
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play an important role in ozone inactivation ofB. subtilis spore
(Cho et al., 2002). In addition to the direct effects of OH radi-
cals on the bacteria cells, it was suggested that hydroxyl
radicals and H2O2might react with peroxidase released from
the bacteria cell as result of the mechanical damage due to
ultrasound cavitation (Jyoti and Pandit, 2003). These reactions
might results in the formation of oxygen radicals, which can
further affect the viability of cells by attacking the cell mem-branes (Jyoti and Pandit, 2003).
TEM observations were performed to investigate the
physical changes induced by high-frequency sonication on
the bacteria cells (Fig. 5). It can be seen that the structure of
these three bacterial species was significantly affected by
sonication. Prior to sonication, the surfaces of bacteria
remained stable, intact and smooth. After sonication, E. aero-
genes were significantly damaged and the cells were
misshapen, malformed, but remained as whole cells,
although the cells loosed their turgor pressure (Fig. 5A2e4).
The effect of high-frequency sonication on E. aerogenescells is
similar to that resulting from the addition of H2O2(Fig. 5B2e4)
confirming again that the main mechanism of inactivation isthe attack of hydroxyl radicals and H2O2 during ultrasound
treatment.However, based on TEM images,the physical effect
of sonication onE. aerogenescell structure (Fig. 5A2e4) seems
to be far greater than addition of H2O2 at an equivalent hy-
droxyl concentration (Fig. 5B2e4). This could be a result of the
mechanical damage due to cavitation in addition to the
decrease in pH. For S. epidermidis, similar results were ob-
tained, with most of the sonicated cells remained whole, and
in some cases leakage of inner contents was also observed
(Fig. 5D2-4). However, in the case ofB. subtilisboth whole cells
and broken parts were observed after sonication (Fig. 5C2e4).
This is an indication thatB. subtiliscan be inactivated through
breakage due to the mechanical effects of ultrasound cavita-tion. This is not surprising as this bacterium was found to be
very sensitive to acoustic cavitation compared to E. aerogenes
andS. epidermidis. This is due to the fact thatB. subtilisare rod
shaped cells and theseare known to be more sensible to break
under acoustic cavitation compared to cocci-shapped ones
(Ahmed and Russell, 1975; Alliger, 1975), and S. epidermidis is
also known to have a thick capsule layer which protects it
against mechanical damage by cavitation (Gao et al., 2014a).
In order to further demonstrate the biocidal effects of
hydroxyl radicals and H2O2 on bacterial cells and estimate
the contribution of mechanical effects on bacterial inacti-
vation by high-frequency ultrasound treatment, t-butanol,
an excellent scavenger of hydroxyl radicals, was added tothe bacteria culture prior to ultrasonication. t-butanol
scavengers hydroxyl radicals following the reaction (Kumar
et al., 2003):
OH CH33COH/H2O CH2CCH32OH (8)
In addition, it is also known that the cavitation bubble
temperature is lowered by the presence oft-butanol at high
frequencies. This is due to the evaporation oft-butanol into
the bubble followed by the accumulation of pyrolysis products
within the bubble (Ashokkumar, 2011). A lower bubble tem-
perature and the presence of pyrolysis products within the
bubble also decreases the amount of OH radicals generated
within cavitation bubbles.
Fig. 6 shows the Log ofsurvivalratio (Log (N/N0)) of different
bacteria ultrasonicated in the presence of different amounts
of t-butanol. Addition of up to 100 mM t-butanol alone to
bacteria cultures does not result in their inactivation (result
not shown). Ultrasonication ofE. aerogenes and S. epidermidis in
the presence of 10 mM t-butanol did not result in the inacti-
vation of these two bacteria (Fig. 6, square and triangle sym-
bols). For E. aerogenes, 4 various concentrations of t-butanolwere added, 0.7 mM, 3 mM, 10 mM, 30 mM and 100 mM. It was
found that E. aerogenes was almost not inactivated when a
power of 50 W was used, and the log reduction increased with
the decreasing concentrations oft-butanol at 62 W. A 2.3-log
reduction was achieved when 0.7 mM t-butanol was added,
and only 0.04 to 0.2 log reductions were achieved for highert-
butanol concentrations. ForS. epidermidissonicated at 62 W, a
0.3-log reduction was achieved when 10 mM t-butanol are
added. However, for B. subtilis, log reductions of 1.6 and 2.0
were achieved in the presence of 10 mM t-butanol and ultra-
sound at 50 W and 62 W, respectively. These values of log
reduction remained smaller than the values of 2.8 and 2.9-log
reduction achieved under similar sonication condition butwith no added t-butanol (Fig. 4B). This would indicate that
although most of the free radicals were scavenged in the
presence of 10 mM t-butanol, the mechanical effects due to
cavitation still played an important role in the inactivation of
B. subtilis. This also confirms TEM observation which clearly
showed that B. subtilis cells do clearly break under high-
frequency ultrasonication.
3.4. Post ultrasonication effects
As stressed early, care needs to be taken in the sampling time
before bacteria counting (Fig. 3) since the effect of high-
frequency ultrasound continues to affect bacteria survivalafter the ultrasonication treatment was ceased. Several
0 20 40 60 80 100-3
-2
-1
0
Concentration of t-butanol (mM)
Log(N/N0)
Fig. 6 e Log of survival ratio (Log (N/N0)) of bacteria as a
function of the concentration oft-butanol.Enterobacter
aerogenesat 50 W (,);Enterobacter aerogenes at 62 W (-);
Bacillus subtilisat 50 W (B);Bacillus subtilisat 62 W (C);
Staphylococcus epidermidisat 50 W (6);Staphylococcus
epidermidisat 62 W (:).
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experiments on E. aerogenes were performed in the present
study in order to investigate theorigin of these effects, and the
results are summarised inTable 1. Firstly as shown inFig. 2,
high-frequency sonication results in a marked decrease in the
pH of the bacteria solution from 6.0 to pH 3.7. To investigate
the effect of pH alone, the pH of PSS solution was adjusted to
pH 3.7 by addition of HCl or Glucono-d-lactone (GDL). These
two acidification methods were used to determine if theinactivation by HCl addition is due to chloride. Two E. aero-
genes suspensions with different initial numbers (w1.49
108 CFU/ml and w1.73 106 CFU/ml) were added to the acid-
ified PSS solutions. Experimental results showed that no
change in the bacteria number wasobserved when leaving the
bacteria under acidic condition (pH 3.7) for 2 h (Table 1). This
experiment allows excluding the contribution of the acidic
environment alone to the inactivation ofE. aerogenes.
The second experiment performed was through the addi-
tion ofE. aerogenesinto pH (3.7 or 6.0) adjusted PSS containing
H2O2. The amount of H2O2 added was 5.88 mM (200 mg/l)
which is 27 times higher than the amount (0.214 mM)
measured in sonicated PSS solution for 20 min at 62 W (Fig. 2).This amount was chosen as it was previously reported that
150 mg/lH2O2 is effective in the inactivation bacteria (Jyoti and
Pandit, 2003). PSS solution was also adjusted to pH 3.7 using
HCl, to take into account the effect of the acidic environment
similar to resulting from sonication. Two E. aerogenes having
different initial numbers of w1.49 108 CFU/ml and
w1.73 106 CFU/ml cultures were added to this PSS solution
and left for 2 h at 4 C temperature. A very slight decrease in
bacteria number (log reduction
-
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that temperature and other conditions used in the published
literature are different. Thus, in light of the different results
reported in the literature on the effect of H2O2on the inacti-
vation of bacteria, the results obtained in the present study
are not unexpected. For instance,it was believed that 100mg/l
H2O2 treatment alone without catalyst stored in dark condi-
tions for 20 min did not inactive E. coli K12 (Paleologou et al.,
2007).Clearly, while H2O2 can inactivate E. aerogenes, the amounts
required to achieve this within 2 h are much higher than that
generated during ultrasonication. It was decided to perform
further experiments as follow. First PSS solution alone was
ultrasonicated at 62 W for 20 min, then the ultrasonication
treatment was stopped. Subsequently, bacteria culture was
added to the ultrasonicated solution and left at 4 C for 2 h.
Bacteria counts showed a log reduction of 2.59 0.23 and
2.75 1.127, forE. aerogenescultures with an initial number of
w1.39 108 CFU/ml and w1.40 106 CFU/ml, respectively
(Table 1). When the same experiments were repeated by
adding bacteria culture into sonicated PSS solutions contain-
ing 10 mM t-butanol, only a very slight log reduction (w0.02)was achieved (Table 1). This finding, in addition to the ex-
periments involving the addition of H2O2 described above,
shows that the most important effect in bacteria inactivation
by high-frequency sonication is due to OH radicals. However,
these OH radicals are expected to undergo a reaction to form
H2O2, and while H2O2can contribute to the inactivation ofE.
aerogenes, its efficiency remains lower than that of the OH
radicals.
3.5. Ultrasound treatment of yeast
High-frequency ultrasound inactivation of yeast, A. pullulans,
was also considered in this study. Two different initial con-centrations (w107 CFU/ml and w105 CFU/ml) ofA. pullulans
were prepared. These suspensions were ultrasonicated for 5,
10, 20, 40, and 60 min at 50 W. Fig. 8 shows the results of
inactivation ofA. pullulans at different initial numbers. The
inactivation of E. aerogenes suspensions at different concen-
trations (w108 CFU/ml, w106 CFU/ml, and w104 CFU/ml) is
also reported in Fig.8 for comparison. Log (N/N0) ofA. pullulans
decreased with a decrease in the initial number. This depen-
dence on the initial number was also previously reported for
this yeast when inactivated by low-frequency ultrasound
treatment (Gao et al., 2014b). Similarly, the inactivation for E.
aerogenesis also found to be dependent on the initial number
(Fig. 8). However, the results show clearly that the yeast A.pullulans is more resistant to high-frequency sonication than
the bacterium E. aerogenes. For instance, one hour ultra-
sonication ofA. pullulansat 50 W resulted in a less than 1 log
reduction for an initial concentration of 4.2 107 CFU/ml, and
less than 2 log reduction for an initial concentration of
3.1 105 CFU/ml. While in the case ofE. aerogenes, under the
same sonication conditions, there was a 3.2-log reduction for
the initial number ofw108 CFU/ml, while 4.4-log reduction for
an initial number of w106 CFU/ml. Note that for the initial
number of w104 CFU/ml, no bacterial colonies were found
after 40 min ultrasonication. It is also worth noting that in the
case of the E. aerogenes, most bacterial inactivation is achieved
during the first 10 min of ultrasound treatment. Increasing
ultrasonication time resulted only in a slight increase in the
reduction of Log (N/N0). For example, for the initial number of
w108 CFU/ml, Log (N/N0) was 2.4 at10 min and decreased to3.2
after 60 min ultrasonication, which probably is due to the
presence of two groups of E. aerogenes cells, with one group
showing more resistance to ultrasonication than the others.The resistance of A. pullulans to ultrasound treatment
compared toE. aerogenesis mainly due to the difference in cell
wall characteristics of these two microorganisms.E. aerogenes
is a gram-negative bacterium and the main components of its
cell wall are protein, polysaccharide, lipid and mucopeptide
(Salton, 1963). The cell wall of gram-negative bacteria is thin,
and mainly composes of 2e7 nm peptidoglycan layer and a
7e8 nm outer membrane (Wang and Chen,2009). The cell wall
of yeast consists mainly of mannoproteins and b-linked glu-
cans (Klis, 1994). It was reported that the cell wall of the
blastospores of mutant A. pullulans B-1 was 60e150 nm
(Gniewosz and Duszkiewicz-Reinhard, 2008). Therefore, based
on the thickness of the cell wall,A. pullulansis expected to bemore resistant to ultrasound treatment than of E. aerogenes.
The light micrographs ofAureobasidium pulluansfor both non-
ultrasonicated and ultrasonicated cells are shown inFig. 9. It
could be seen that some cell envelopes (see arrows) are pre-
sent in both sonicated and non-sonicated samples, and this
could be a result of the damage to the yeast cells resulting in
the leaking of their inner contents.
4. Conclusions
The effects of high-frequency ultrasound on three species of
bacteriaE. aerogenes, B. subtilis and S. epidermidis and a yeast
0 10 20 30 40 50 60
-4
-3
-2
-1
0
Ultrasound time (min)
Log(N
/N0)
Fig. 8 e Log of survival ratio (Log (N/N0)) for ultrasonicated
yeast and bacteria as a function of ultrasonication time in
different initial numbers. The initial cell numbers ofAureobasidium pullulansare w4.2 3 107 CFU/ml (-),
w3.1 3105 CFU/ml (C). The initial cell numbers of
Enterobacter aerogenesare w1.5 3108 CFU/ml (,),
w1.7 3106 CFU/ml (B), w1.5 3104 CFU/ml (6). Error bars
correspond to standard deviation.
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strain, A. pullulans, were investigated. These bacteria were
chosen as they have different cell wall type, size and shape.
These bacteria were also sonicated at different growth phases,
namely the exponential and stationary phases. The study
found that high-frequency ultrasound, at high applied powers
or longer treatment times, is efficient to inactivate bacteria in
both growth phases. Ultrasonication resulted in the inacti-
vated of more than 99% of the bacteria. In contrast, the yeast
A. pullulanswas found to be very resistant to high-frequency
ultrasound treatment. However, high-frequency ultra-sonication was also found to be less efficient at lower applied
powers or shorter treatment times. Thus, further studies are
needed to determine energy requirements for the imple-
mentation of high-frequency ultrasound technology for bac-
teria inactivation in an industrial environment.
It was found from this study that the main mechanism of
the high-frequencyultrasonic inactivation of bacteria is due to
sonochemical effects related to the generation of hydroxyl
radicals and hydrogen peroxide. This was confirmed by TEM
observation which showed that most of the bacteria cells
retained their physical integrity. However, TEM observation
also showed that the rod-shaped B. subtilis cells were sus-
ceptible to mechanical damage due to cavitation. It was also
found that there was a post-sonication effect on the bacteria
cultures. In other words, the longer the residence time of the
bacteria in the ultrasonicated medium, that is after sonication
treatment is stopped, the higher the number of inactivated
bacteria cells. Overall this study indicates that high-frequency
ultrasound presenta potential for the inactivation of microbes
in contaminated water, and thus can be used in wastewater
treatment.
Acknowledgements
S. Gao is supported by a New Zealand China Food Safety
Scholarship, funded by the New Zealand Ministry of Foreign
Affair and Trade. Funding for the purchase of the high-
frequency unit is covered by a CMI-DB breweries grant.
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