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

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Inactivation of bacteria and yeast using highfrequency 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 Zealand School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand c Institute of Food and Agricultural Standardization, China National Institute of Standardization, Beijing 10088, China d School of Chemistry, University of Melbourne, VIC 3010, Australia b

article info

abstract

Article history:

High-frequency (850 kHz) ultrasound was used to inactivate bacteria and yeast at different

Received 30 September 2013

growth phases under controlled temperature conditions. Three species of bacteria, Enter-

Received in revised form

obacter aerogenes, Bacillus subtilis and Staphylococcus epidermidis as well as a yeast, Aur-

11 April 2014

eobasidium pullulans were considered. The study shows that high-frequency ultrasound is

Accepted 19 April 2014

highly efficient in inactivating the bacteria in both their exponential and stationary growth

Available online 1 May 2014

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

Keywords:

free radicals and H2O2. The rod-shaped bacterium B. subtilis was also found to be sensitive

High-frequency ultrasound

to the mechanical effects of acoustic cavitation. The study showed that the inactivation

Bacteria inactivation

process continued even after ultrasonic processing cessed due to the presence of H2O2,

Yeast

generated during acoustic cavitation. Compared to bacteria, the yeast A. pullulans was

Enterobacter aerogenes

found to be more resistant to high-frequency ultrasound treatment.

Bacillus subtilis

ª 2014 Elsevier Ltd. All rights reserved.

Staphylococcus epidermidis Aureobasidium pullulans

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

* Corresponding author. Tel.: þ64 9 9239676; fax: þ64 9 3737422. E-mail address: [email protected] (Y. Hemar). http://dx.doi.org/10.1016/j.watres.2014.04.038 0043-1354/ª 2014 Elsevier Ltd. All rights reserved.

eliminate microorganisms in water through chemical oxidation (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 inactivated by chlorination as they form clusters (Phull et al., 1997). As a result, the use of ultrasound treatment can be considered

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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 reported 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 suggested to be due to mechanical effects and sonochemical reactions 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 ultrasound 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 cavities, and the energy released can damage bacteria (Collis et al., 2010; Joyce et al., 2003a; Mason et al., 2003, 2005; O’Donnell et al., 2010; Soria and Villamiel, 2010; Wu and Nyborg, 2008). Although cavitation can be produced during both lowfrequency 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 frequency higher than 100 kHz [2, 9e11]. For high-frequency ultrasound, 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 chemical bonds, 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 membrane, and the recombination of these radicals into hydrogen peroxide, a well-known bactericide, also contributes to bacteria 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 the effects 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 inactivation 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 organism (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 microbiological 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; Urzi et al., 1999). E. aerogenes is a gram-negative rod-shaped bacterium from the family of Enterobacteriaceae and the size range of these bacteria are 0.3e1.0 mm  1.0e6.0 mm (Imhoff, 2005). B. subtilis is a grampositive 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. pullulans is 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 studied widely for the disinfection of wastewater, involving microbes 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 considered in 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 effect of high-frequency ultrasound and vancomycin on S. epidermidis 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 and A. 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 purchased 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, and A. pullulans were obtained 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 then transferred into 100 ml fresh Nutrient Broth to be incubated under the same conditions. The exponential 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 incubation for E. aerogenes, B. subtilis and S. epidermidis, respectively, and the bacteria suspensions at stationary phase were obtained after 20 h incubation. A. pullulans was incubated at 28  C, and the yeast suspension to be ultrasonicated 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 nutrients 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 centrifuged at 3000 g for 10 min at 4  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 temperature during ultrasonication. Under the ultrasound conditions used in this study, the working temperature in the vessel never exceeds 20  C. All ultrasound treatments were performed at least in duplicate.

95

The ultrasound power P was determined using the calorimetric method (Kikuchi and Uchida, 2011; Koda et al., 2003): P ¼ mCp

  DT Dt

(1)

where Cp (¼4.18 J/(g K)) is the specific heat capacity of water, m is the mass of ultrasonicated water, DT is the increase in the temperature, and Dt is 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 performed 2 h after ultrasound treatment. For each sample, serial 1:10 dilutions from 100 to 106 times depending on the initial number of microorganisms 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 incubated at 37  C overnight for the bacteria and at 28  C for 24e36 h for the yeast. The countable number (15e150) of colonies for each section was counted under a light microscope. The original bacterial number was calculated using: N ¼ 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 volume of 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 measurement of the yield of H2O2 by using a UVeVis absorption

Fig. 1 e 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 iodide reagents before measuring the absorbance. The concentration of H2O2 (c, M) can be calculated by the following equation: A c¼ 3 εl

(3)

where A is absorbance value at 353 nm, ε 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 suspensions 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 with S. epidermidis was 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

(4)

(5)

The iodide method is commonly used to quantify the concentration of hydrogen peroxide produced during ultrasonication (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 I 3 complex (Reaction 7), which can be detected by UV measurements at 353 nm. H2O2 þ 2I / I2 þ 2OH

(6)

I2 þ I /I 3

(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 H2O2 can be clearly seen for ultrasound power higher than 10 W. These results are in agreement with previous studies which showed that high-frequency ultrasonication results in an increase of H2O2 with the ultrasonication power (Kanthale et al., 2008). It should be noted that post ultrasonication, the amount of the H2O2 slowly decreased with time (results not shown). The increase in H2O2 with 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.

As mentioned previously, acoustic cavitation induces mechanical effects and sonochemical reactions such as the formation of hydroxyl radicals during high-frequency ultrasonication. For low-frequency high-power ultrasound treatment of bacteria, inactivation was mainly due to mechanical effects induced by acoustic cavitation (Gao et al., 2014a,b). However in the case of high-frequency ultrasonication, 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 hydroxyl 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 radicals and H2O2 (Reactions (4) and (5)) (Dai et al., 2006; Olvera et al., 2008; Thangavadivel et al., 2009): H2O þ )))) / OH þ H

2OH / H2O2

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 H2O2 change with time post ultrasonication as indicated

Fig. 2 e Hydrogen peroxide (-) produced by highfrequency 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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inactivated. This result is very important as it demonstrates clearly that 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 effects of high-frequency (850 kHz) ultrasound treatments of different bacteria at different growth phases were investigated. 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

Log(N/N0)

-1 -2 -3 -4 -5 0

10

20

30

40

50

60

70

40

50

60

70

40

50

60

70

(B)

0 -1 -2 -3 -4

0

-5

-1

0

-2 -3

10

20

0

-4

30

(C)

-1

-5

Log(N/N0)

Log (N/N 0 )

(A)

0

Log(N/N0)

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 for 20 min, 62 W for 10 min and at 62 W for 20 min. A nonultrasonicated E. aerogenes suspension was also used as a control. The ultrasound treated E. aerogenes suspensions 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 sonicated E. 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 was also noticeable for suspensions 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

-6 -7 -8 0

50

100

150

200

250

300

Sampling time (min) Fig. 3 e Log of survival ratio (Log (N/N0)) of Enterobacter aerogenes as 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.

-2 -3 -4 -5 0

10

20

30

Ultrasound Power (W) 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 ultrasonication for E. aerogenes, B. subtilis and S. epidermidis, respectively, in the stationary phase. Moreover, there was no marked difference for the inactivation ratio between the bacteria at exponential phase and at stationary phase. In addition, there was no obvious effect on the bacterial viability at lower power ultrasound treatment (at 9 W or 15 W), which is likely due to the lower concentration of hydroxyl radicals and hydrogen peroxide produced at lower powers as shown in Fig. 2.

It is likely that the inactivation of the bacteria is due to the generated hydroxyl radicals and H2O2 directly attacking bacterial cells. The formation of free radicals during ultrasonication 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, hydroxyl radicals 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 disintegrated 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 epidermidis before (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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OH þ ðCH3 Þ3 COH/H2 O þ CH2 CðCH3 Þ2 OH

(8)

In addition, it is also known that the cavitation bubble temperature is lowered by the presence of t-butanol at high frequencies. This is due to the evaporation of t-butanol into the bubble followed by the accumulation of pyrolysis products within the bubble (Ashokkumar, 2011). A lower bubble temperature 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 of survival ratio (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 of E. aerogenes and S. epidermidis in the presence of 10 mM t-butanol did not result in the inactivation of these two bacteria (Fig. 6, square and triangle symbols). For E. aerogenes, 4 various concentrations of t-butanol were 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 of t-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 higher tbutanol concentrations. For S. epidermidis sonicated 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 ultrasound 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 but with 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 highfrequency 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 highfrequency ultrasound continues to affect bacteria survival after the ultrasonication treatment was ceased. Several

0

Log(N/N0)

play an important role in ozone inactivation of B. subtilis spore (Cho et al., 2002). In addition to the direct effects of OH radicals on the bacteria cells, it was suggested that hydroxyl radicals and H2O2 might 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 membranes (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. aerogenes 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. aerogenes cells is similar to that resulting from the addition of H2O2 (Fig. 5B2e4) confirming again that the main mechanism of inactivation is the attack of hydroxyl radicals and H2O2 during ultrasound treatment. However, based on TEM images, the physical effect of sonication on E. aerogenes cell structure (Fig. 5A2e4) seems to be far greater than addition of H2O2 at an equivalent hydroxyl 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 obtained, 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 of B. subtilis both whole cells and broken parts were observed after sonication (Fig. 5C2e4). This is an indication that B. subtilis can be inactivated through breakage due to the mechanical effects of ultrasound cavitation. This is not surprising as this bacterium was found to be very sensitive to acoustic cavitation compared to E. aerogenes and S. epidermidis. This is due to the fact that B. subtilis are rod shaped cells and these are 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 inactivation by high-frequency ultrasound treatment, t-butanol, an excellent scavenger of hydroxyl radicals, was added to the bacteria culture prior to ultrasonication. t-butanol scavengers hydroxyl radicals following the reaction (Kumar et al., 2003):

-1

-2

-3

0

20

40

60

80

100

Concentration of t-butanol (mM) Fig. 6 e Log of survival ratio (Log (N/N0)) of bacteria as a function of the concentration of t-butanol. Enterobacter aerogenes at 50 W (,); Enterobacter aerogenes at 62 W (-); Bacillus subtilis at 50 W (B); Bacillus subtilis at 62 W (C); Staphylococcus epidermidis at 50 W (6); Staphylococcus epidermidis at 62 W (:).

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experiments on E. aerogenes were performed in the present study in order to investigate the origin of these effects, and the results are summarised in Table 1. Firstly as shown in Fig. 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 the inactivation by HCl addition is due to chloride. Two E. aerogenes suspensions with different initial numbers (w1.49  108 CFU/ml and w1.73  106 CFU/ml) were added to the acidified PSS solutions. Experimental results showed that no change in the bacteria number was observed 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 of E. aerogenes. The second experiment performed was through the addition of E. aerogenes into 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/l H2O2 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 < 0.1) was observed (Table 1). Note that when bacteria was left for 2 h in a PSS solution containing the same amount of H2O2 but having a pH 6.0, similar results on the number of bacteria inactivated were obtained. However, when the bacteria culture was added to PSS solution with 5.88 mM H2O2 having a pH 3.7 and left for 24 h, the number of bacteria decreased markedly and a log

reduction of 1.66 was obtained. This also shows that the effect of H2O2 is time dependent and that the longer is the residence time of the bacteria in a medium containing H2O2 the higher is the extent of their inactivation. A possible way to increase the efficiency of H2O2 to inactivate bacteria is by increasing the amount added rather than increasing the residence time of the bacteria culture. Fig. 7 shows the Log of survival ratio (Log (N/N0)) for E. aerogenes as a function of residence time of H2O2 in a PSS solution (pH 6.0). It can be seen that for 0.214 mM and 5.88 mM H2O2, no marked change in the Log (N/N0) is observed for a residence times of up to 2 h. This confirms the results reported in Table 1 for PSS solution containing 5.88 mM under acidic and non-acidic conditions. However, when the H2O2 is increased to 200 mM, log (N/N0) decreases markedly with the increase in residence time to reach a value of 3.5 after incubation for 2 h. As a well-known powerful oxidant, H2O2 is widely used as a biocide and it is believed that the core of the oxidation effect of hydrogen peroxide is due to hydroxyl radicals generated during the decomposition of H2O2 (Jyoti and Pandit, 2003; Petri et al., 2011; Wolfe et al., 1989). Previous studies showed that 1% (294 mM) to 3% (882 mM) H2O2 have been used as dental and contact lens disinfectant solutions (Hughes and Kilvington, 2001; Walker et al., 2003), and 1% (294 mM) was used to decontaminate apples (Sapers and Sites, 2003). It was observed that about 3-log reduction can be achieved when E. coli K12 is exposed to 50 mM H2O2 at 37  C for 15 min (Imlay and Linn, 1986). It was also reported that 5 mg/l H2O2 (0.147 mM) treatment for 15 min caused 13%, 9% and 9% reduction in bore well water for total coliforms, fecal coliforms and fecal streptococci individually, while 95%, 96% and 88% reduction achieved, respectively when a combination of ultrasonic bath and 5 mg/l H2O2 (0.147 mM) treatments are used (Jyoti and Pandit, 2003). Clearly, the minimal amount of H2O2 to inactivate bacteria is still not well known, and it might depend on the bacteria species. Further, it needs to be clarified

Table 1 e Log reduction of bacteria (Enterobacter aerogenes) in different pH buffer.

Acidic PSS (by HCl) Acidic PSS (by HCl) Acidic PSS (by GDL) Acidic PSS (by GDL) Adding H2O2 in acidic PSS Adding H2O2 in acidic PSS Adding H2O2 in PSS* Adding H2O2 in acidic PSS* Ultrasonicated PSS Ultrasonicated PSS Ultrasonicated PSS þ t-butanol Ultrasonicated PSS þ t-butanol

pH

3.7 3.7 3.7 3.7 3.7 3.7 6.0 3.7 3.7 3.7 6.3

Initial Log Standard number reduction deviation (CFU/ml) 1.49  1.73  1.49  1.73  1.46  1.26  1.41  1.41  1.39  1.40  1.55 

8

10 106 108 106 108 106 108 108 108 106 108

0.00 0.01 0.04 0.07 0.00 0.08 0.08 1.66 2.59 2.75 0.02

0.05 0.01 0.03 0.03 0.04 0.06 0.07 0.84 0.23 1.13 0.01

6.3 1.18  106

0.06

0.03

Note: (1) The reaction time without sonication of bacteria in buffers was 2 h except labelled (*: 24 h). (2) GDL stands for Glucono-dlactone. (3) Added H2O2: 5.88 mM (4) Ultrasonication conditions: 850 kHz, 62 W, 20 min.

-1

Log(N/N0)

Buffer

0

-2

-3

-4

0

20

40 60 80 Sampling time (min)

100

120

Fig. 7 e Log reduction of Enterobacter aerogenes as a function of reaction time after addition of different H2O2 amounts: 0.214 mM (-), 5.88 mM (C), 200 mM (:). Note that ultrasonication at 62 W for 20 min produces 0.214 mM H2O2. Error bars correspond to standard deviations.

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3.5.

Ultrasound treatment of yeast

High-frequency ultrasound inactivation of yeast, A. pullulans, was also considered in this study. Two different initial concentrations (w107 CFU/ml and w105 CFU/ml) of A. 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 of A. pullulans at different initial numbers. The inactivation of E. aerogenes suspensions at different concentrations (w108 CFU/ml, w106 CFU/ml, and w104 CFU/ml) is also reported in Fig. 8 for comparison. Log (N/N0) of A. pullulans decreased with a decrease in the initial number. This dependence 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. aerogenes is 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 ultrasonication of A. pullulans at 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 of E. aerogenes, under the same sonication conditions, there was a 3.2-log reduction for the initial number of w108 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

0

-1

Log(N/N0)

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 H2O2 on the inactivation of bacteria, the results obtained in the present study are not unexpected. For instance, it was believed that 100 mg/l H2O2 treatment alone without catalyst stored in dark conditions 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, for E. aerogenes cultures 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 containing 10 mM t-butanol, only a very slight log reduction (w0.02) was achieved (Table 1). This finding, in addition to the experiments 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 H2O2 can contribute to the inactivation of E. aerogenes, its efficiency remains lower than that of the OH radicals.

-2

-3

-4 0

10

20

30

40

50

60

Ultrasound time (min) 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 of Aureobasidium pullulans are w4.2 3 107 CFU/ml (-), w3.1 3 105 CFU/ml (C). The initial cell numbers of Enterobacter aerogenes are w1.5 3 108 CFU/ml (,), w1.7 3 106 CFU/ml (B), w1.5 3 104 CFU/ml (6). Error bars correspond to standard deviation.

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 at 10 min and decreased to 3.2 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 to E. aerogenes is 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 glucans (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. pullulans is expected to be more resistant to ultrasound treatment than of E. aerogenes. The light micrographs of Aureobasidium pulluans for both nonultrasonicated and ultrasonicated cells are shown in Fig. 9. It could be seen that some cell envelopes (see arrows) are present 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 bacteria E. aerogenes, B. subtilis and S. epidermidis and a yeast

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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 present a 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 highfrequency unit is covered by a CMI-DB breweries grant.

references

Fig. 9 e Light micrographs of Aureobasidium pullulans (A) before ultrasound treatment and (B) after ultrasound treatment for 60 min at 62 W.

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 inactivated of more than 99% of the bacteria. In contrast, the yeast A. pullulans was found to be very resistant to high-frequency ultrasound treatment. However, high-frequency ultrasonication 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 implementation of high-frequency ultrasound technology for bacteria inactivation in an industrial environment. It was found from this study that the main mechanism of the high-frequency ultrasonic 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 susceptible to mechanical damage due to cavitation. It was also

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