inactivation of bacteria and yeast using high frequency ultrasound treatment.pdf

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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

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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 (:).

w a t e r r e s e a r c h 6 0 ( 2 0 1 4 ) 9 3 e1 0 4 99


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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.

w a t e r r e s e a r c h 6 0 ( 2 0 1 4 ) 9 3 e1 0 4 101


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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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