Combined Effect of Ultrasound and Ozone on Bacteria in Water

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The combined effect of ultrasound and ozone on bacteria in water Amna Mohammed Al-hashimi, Timothy J. Mason, and Eadaoin Maria Joyce Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5045437 • Publication Date (Web): 16 May 2015 Downloaded from http://pubs.acs.org on May 18, 2015

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Environmental Science & Technology

The combined effect of ultrasound and ozone on bacteria in water

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Amna M. Al-Hashimi, Timothy J. Mason and Eadaoin M. Joyce*

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The Sonochemistry Centre, Coventry University, Faculty of Health and Life Sciences, Priory Street, Coventry, UK

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Tel: +44 2476888075; Fax: +44 2476 888173; *Corresponding author email:[email protected]

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Keyword: Ozone,ultrasound,water treatment, flow cytometry, Escherichia coli

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*Corresponding author e-mail:[email protected]

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Abstract ........................................................................................................................................... 2

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Introduction ..................................................................................................................................... 3

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Material and methods ...................................................................................................................... 5

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Preparation and viability assessment of bacterial suspensions ................................................... 5

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Ultrasound and ozone system (USO3) ........................................................................................ 6

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Transmission electron microscopy ............................................................................................. 7

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Results ........................................................................................................................................... 10

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Measurement of dissolved ozone in water ................................................................................ 10

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Effect of ultrasound on the viability of Escherichia coliusing theUSO3 system. ..................... 11

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Effect of ozone on 75L Escherichia coli treated with the USO3 system for 16 minutes ......... 13

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Combined effect of ultrasound and ozone on the viability of Escherichia coli treated using the USO3 system ............................................................................................................................. 14

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Discussion ..................................................................................................................................... 16

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Acknowledgment .......................................................................................................................... 21

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References ..................................................................................................................................... 21

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ACS Paragon Plus Environment

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Abstract

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the biological disinfection of water on a large-scale application using viable plate counts and

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flow cytometry.

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Methods and results: Escherichia coli B bacteria in saline suspension was treated using a

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commercially available combined ultrasound and ozone system (USO3 - USS, Ultrasonic

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Systems Gmbh, Germany) for 16 minutes. Two analytical methods were used to assess the

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results in terms of live and dead cells in the bulk liquid: standard viable plate counting recorded

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in terms of Colony Forming Units (CFU) per ml and flow cytometry (FCM). In the latter case 1

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ml bacterial suspension was stained simultaneously with the fluorescent stains SYTO9 and

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Propidium Iodide (PI). Transmission electron microscopy was used to generate images

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identifying the biological effects of different treatments using ultrasound and ozone on bacterial

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cell walls. Results demonstrated that treatment with ozone alone (1mg/l) resulted in a significant

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reduction (93%) in the number of live cells after 16 minutes treatment whereas ultrasound alone

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showed only a small reduction (24%). However a combination of ozone and ultrasound showed a

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synergistic effect and enhanced the inactivation to 99%after 4 minutes.

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Conclusion: A combined ultrasound and ozone treatment of bacterial suspensions using a

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commercial system (USS, Ultrasonic Systems Gmbh) affords a promising method for water

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disinfection that is better than treatment using either method alone. Standard viable plate count

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analysis is normally used to assess the effectiveness of disinfection treatments; however flow

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cytometry proved to be a more sensitive method to determine the actual effects in terms of not

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only live and dead cells but also damaged cells. This type of analysis (cell damage) is difficult if

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not impossible to achieve using traditional plate counting methodology.

Aim of the study: To assess the synergetic effect of combined ultrasound and ozone treatment on


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Introduction

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replacement for chlorination in that it reduces disinfection by-products [1]. It is an unstable tri-

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oxygen molecule which easily releases oxygen atoms in aqueous solutions which subsequently

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reacts with hydrogen ions in water leading to increased levels of hydroxyl radicals (OH●) in the

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medium [2]. Joret showed that ozone had lethal effects on a wide range of microbes, such as

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bacteria, viruses, parasites, Candida, polio virus and Mycobacterium through oxidizing the cell

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membrane resulting in cell disruption [3]. Disinfection by ozone is strongly dependent on water

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characteristics such as dissolved organic carbon (DOC), pH, temperature and bromide

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concentrations [4][5].

Ozone is an important disinfection gas for drinking water treatment and is a suitable

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Over the last 20 years researchers and engineers have found that a combination of two or more

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disinfection methods involving oxidation (Advanced Oxidation Processes - AOPs) is very

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effective in water purification. AOPs help to improve water quality and reduce treatment costs

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[6]. Combinations of ultrasound with other oxidants such as ozone, chlorine and hydrogen

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peroxide (H2O2) have been investigated [4]. These studies concluded that employing a

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combination of ozone and ultrasound treatments in the secondary stage of wastewater treatment

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resulted in reductions in the required contact time to inactivate microorganisms.

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The collapse of acoustic cavitation bubbles generated during the application of ultrasound to

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aqueous systems affords several routes to disinfection [7][8]. Cavitation bubble collapse creates

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a “hot spot” where extremely high temperatures, pressures and free radicals form [9]. The

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possible effects of sonication on microorganisms are:

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

High temperatures in and around the collapsing bubble resulting in enzyme denaturation


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

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High hydro-mechanical pressures generated on bubble collapse resulting in shear forces and liquid jets which lyse cells



Production of a highly oxidative species of OH● and H2O2from the hot spots which can attack and weaken cell membranes by attacking their chemical structure [10] [11].

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The impact of ultrasound on bacteria has been reported in previous studies that focus on cell

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inactivation, the release of cell contents and the production of a temporary permeability of cell

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walls. In addition, the effect of ultrasound on inactivation of microorganisms using varying

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treatment times for many pure cultures of different bacteria, yeast, fungi and viruses has been

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reported [12][13].

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Microorganism inactivation has also been investigated using a combination of ultrasound with

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ozone or UV radiation [14][15][16][17], high pressures and temperatures, or different ultrasonic

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parameters [18][19].

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Previous investigations of combined treatments with ozone and ultrasound have indicated that

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the synergistic effect of sonication and ozone significantly increases bacterial inactivation rates.

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Jyoti and Pandit indicated that microbial damage resulting from hydro-mechanical forces is

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greater than sonochemical effects [20]. It was reported that the combination of hydrodynamic

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acoustic cavitation and ozone reduced power consumption, in addition to reducing the formation

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of disinfection by-products (DBP) during treatment [21].

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Jyoti and Pandit used the viable plate count (VPC) analysis method to assess the effect of

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acoustic cavitation and ozone on microbial disruption [22]. However, culture based methods are

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time consuming and do not identify sub-lethally damaged cells. Such cells retain some metabolic

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activity but are unable to produce a viable colony on solid growth media (agar). This can occur


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when bacteria undergo stress but are not completely inactivated or killed by water treatment

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methods such as chlorination. However, if sufficient numbers of these organisms are ingested

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they have the potential to cause illness [23]. Therefore, there is an increasing interest in finding

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an analytical technique which can yield more information on the physiological status of bacteria

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than can be deduced from standard culture methods. Additionally, different environmental

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conditions can affect the viable counting method [24]. Various characteristics must be

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considered when evaluating microbial viability which includes membrane integrity, cell growth

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and metabolic activity; however membrane integrity has received the majority of research [25].

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Flow cytometry (FCM) is capable of quantifying features of cells primarily by visual means.

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FCM records measurements from individual cells but can process thousands of cells (10,000

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events) in seconds. In conjunction with fluorescent staining it can provide information relating to

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the estimation and discrimination of different physiological characteristics of cells [24].The aim

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of this study was to evaluate the application of FCM with dual staining for a rapid, accurate and

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reliable assessment of cell viability after ultrasound, ozone and combined treatments using the

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USS system. The results were compared with those obtained using a traditional CFU culture

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method.

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Material and methods Preparation and viability assessment of bacterial suspensions

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A stock suspension of Escherichia coli was prepared according to [26]. 1 ml of bacterial

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suspension was taken after 0, 2, 4, 8 and 16 minutes treatment and added to 9 ml of 0.9% saline

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(9 g/L sigma Aldrich Ltd.) containing sodium thiosulfate Na2S2O3 (2 g/l) to neutralise any

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residual ozone in the sample. Samples were serially diluted to obtain 2g/l [14]. Each dilution was


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cultured on nutrient agar plates in triplicates and incubated at 37°C for 24 hours. The results

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from plate counts were converted into CFU/ml:

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CFU’s/ml = No. of colonies * volume of cultured sample * dilution of sample

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Ultrasound and ozone system (USO 3 )

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A large-scale water treatment device from Ultrasonic Systems Gmbh, Germany, was employed

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to deliver ultrasound and ozone for water treatment using a technology known as USO3. The

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system consisted of an ozone generator, 20 ultrasonic transducers at maximum power setting

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(100 Watt each/612 kHz), a mixer and external pump that was capable of treating up to a

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maximum capacity of 4 m3/h = 66.67 L/min. Internally, the system consisted of long steel tubes

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with a diameter of 108.3 mm, L = 3.02 m, V = 28 L = 0.028 m3), retention time: 0.8 min. The

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bacterial suspension was pumped into the USO3 kit using an external pump (flow rate 35 L/min).

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Experiments were performed at a constant flow rate of 35 L/min, delivering a constant ozone

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supply capable of generating a concentration which accumulated to 1 mg/L. The aqueous

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suspension of the bacteria was treated at this flow rate in a recycling system for 16 minutes with

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pulsed ultrasonic treatment on for 5 seconds and off for 5 seconds. The ozone concentration was

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measured using the indigo colorimetric method [27].

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Transmission electron microscopy

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A transmission electron microscope TEM was used to provide high resolution images, (2010F,

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JEOL Ltd., Japan). The instrument used a 200kV field emission gun instrument in cryo-

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conditions and was fitted with a GatanUltraScan™ 4000 camera which provided the spatial


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resolution necessary for detailed biological structural analysis and very high magnification of

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samples down to 0.5nm with three dimensional imaging.

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Experiment design

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75 L of Escherichia coli bacterial suspension (OD 0.01, 2x105 cell/ml) was treated with

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ultrasound alone, ozone alone (1mg/l, power 160 W, 10% gas flow) and a combination of

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ultrasound and ozone for 16 minutes. Samples were taken after 0, 2, 4, 8 and 16 minutes

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treatment and the residual ozone in sample was neutralised using 2% sodium thiosulfate (Sigma

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Aldrich Ltd.) in sterile saline (0.9% NaCl Sigma Aldrich Ltd.). An in-built cooling system was

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used to maintain sample temperature below 25ºC. Samples were analysed by viable plate counts

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to measure CFU/ml and flow cytometry. All data was analysed using ANOVA test.

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Bacterial staining and flow cytometry

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The use of double staining techniques involving nucleic acid stains (SYTO9) (green

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fluorescence) and Propidium Iodide (PI) (red fluorescence) provide a valuable method for the

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estimation of bacteria viability [185]. The fluorescent probes/stains and counting kit for flow

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cytometry were purchased from Invitrogen (LIVE/DEAD® BaclightTM bacterial viability and

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counting kit) which consisted of two stains SYTO9 (nucleic acid stain) at a dose of 200 µl (3.34

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mM in DMSO) specific for live cells, Propidium Iodide (PI) at a dose of 200 µl (20 mM in

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DMSO) for dead cells. The calibration beads were microsphere standard beads 6.0 µm diameter

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with a concentration of 1×108 beads/ml in deionised water (10ml) containing 2 mM sodium

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azide.

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Flow cytometry measures several parameters at the same time for each cell:


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Low angle forward scatter intensity (FSC) is proportional to cell diameter.

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Orthogonal (90º) scatter intensity (SSC) relates to the quantity of granular structures within the

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cell. Fluorescence intensities at several wavelengths can be observed using different fluorescence

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channels.

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Estimation of cell integrity or viability using flow cytometry

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500 µl bacterial suspensions were collected in an Eppendorf tube (1.5 ml). Samples were

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simultaneously stained with SYTO9 and Propidium Iodide (PI). Fluorochromes were added

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according to the manufacturer’s instructions. Dyes were purchased as a BaclightTM bacterial

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viability and counting kit from Invitrogen Ltd. 1.5 µl of fluorescent stain was added to each

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sample, vortexed and analysed after 10-30 minutes incubation at room temperature. Excitation of

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SYTO9 emitted green fluorescence at 525-550 nm and excitation of PI at 536nm produced red

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fluorescence at 600nm. Viable cells (with intact membranes) are only permeable to SYTO9 but

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not PI; hence viable cells emit green fluorescence. In contrast, dead cells (with permeabilised

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membranes) were stained with PI and emit red fluorescence. BD FACS Calibur flow cytometry

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was optimised by FACS COMP standard fluorescence beads (2 µm) to verify the instrument

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performance. Bacterial populations were positioned so that they were all located in scale on a

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FSC V’s SSC plot. For fluorescence measurements, FL1, FL2 and FL3 voltages were adjusted to

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place the unstained populations in the lower left quadrant of two parameter plots. Flow

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cytometry instruments settings were as follows: threshold (FSC), FSC (E02), PMT 4.75

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(logarithmic amplification (LA), SSC-597 V (LA), FL1 617/550 V (LA), FL2 531 V (LA) and

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FL3 623 V (LA). 10,000 cells were acquired per acquisition and bacterial populations were gated

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using a combination of FSC, SSC, live/dead cells and discriminated using FL1 V’s FL3. Live (3

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hour bacterial culture) and dead (heating bacterial cells to 75°C for 30 minutes) controls were


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also undertaken to calibrate results. To ensure continuity of results all experiments were run for a

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total of 16 minutes.

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Effect of ultrasound and ozone separately a nd in combination on the biological structure of bacterial cell walls using CRYO TEM analysis

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Escherichia coli bacterial suspension was placed in a suitable container and treated with

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ultrasound alone ozone alone and a combination of ultrasound and ozone for 16 minutes using

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the USO3 system. Samples were taken at 0, 2, 4, 8 and 16 minutes and analysed using TEM

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microscopy. To determine the percentage inactivation by TEM, a protocol was followed where

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intact cells were considered to be robust and cells displaying a loss of cell integrity were

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considered dead. For each TEM calculation 100 cells within a grid were observed following each

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treatment. Cells which appear to show damage were then converted to a percentage.

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Supporting Information

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Figure 1 Schematic diagram of the USO3 system taken from the manual provided by USS, (20

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ultrasonic transducers at maximum power setting (100 Watt each/612 kHz)

Output

Input

205 Ultrasonic transducer

Opti-mixer


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Measurement of dissolved ozone in water

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NaCl (0.9 g/l) was added to 75 L of fresh water to convert the water to saline which was

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circulated through the ultrasound and ozone (USO3) system for 16 minutes. 50ml of sample was

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collected and analysed using the UV-Vis spectrophotometer (Shimadzu, UK 5030). Results

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illustrate that the absorbance of water samples at 254 nm increased over 16 minutes treatment,

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which is due to the accumulation of ozone in water over 16 minutes treatment (Figure 2).

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Figure 2 Accumulation of residual ozone in 75 L water using the ultrasound and ozone system

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over 16 minutes analysed using UV-Vis spectrophotometry

Control

Ozone (ppm)

Abs.

Time [min]

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Results

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Effect of ultrasound on the viability of Escherichia coliusing theUSO 3 system.

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A single bacterial suspension of Escherichia coli was treated with ultrasound for 16 minutes.

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Results illustrate a small inactivation effect of 24% on the number of live cells over 16 minutes

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treatment compared to controls (Figure 3). This is in accord with other results obtained at a


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laboratory scale using higher frequencies [26] [28]. 512 kHz was used to treat different types of

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bacteria for 15 and 60 minutes which achieved less than 1 log reduction at the end of the

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treatment time.

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Figure 3 Effect of ultrasound on the viability of Escherichia coli, (OD 0.01, 2x105 cell/ml) using

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the USO3 system for 16 minutes analysed using viable plate counts Control

CFU/ml

US

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Time [min]

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The bacteria were also analysed by flow cytometry using the Invitrogen BaclightTM bacterial

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viability and counting kit. The dot plots illustrated in Figure 4 represent bacterial populations

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that are divided into two populations; live cells appearing in the lower right (LR) quadrant and

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dead cells in the lower left (LL) quadrant. Flow cytometry data illustrates small reductions of

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26% in the number of live cells after 16 minutes treatment. Flow cytometry data supports viable

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plate count results, but some cells were observed in the live cells position after treatment.

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Although there were still some cells observed in the position expected for live cells the majority

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of cellular material after treatment appeared in an intermediate position between that for live


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cells and that for dead cells. This is where one would normally find viable but non culturable

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(VBNC) cells. Cell wall fragments may up take some of the background fluorescent stain.

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Figure 4 Effect of ultrasound on the viability of Escherichia coli treated with the USO3 system

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for 16 minutes analysed using flow cytometry

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Control

16 min

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Effect of ozone on 75L Escherichia coli treated with theUSO 3 system for 16 minutes

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A single bacterial suspension of Escherichia coli was treated with ozone using the USO3

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system for 16 minutes. Results demonstrate a 93% reduction in number of live E. coli cells over

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16 minutes treatment time (Figure 5). The results obtained for reductions in E. coli are in accord

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with those of other researchers who achieved 1 log reduction for E. coli [30].

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Figure 5 Effect of ozone on Escherichia coli using the USO3 system for 16 minutes analysed

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using viable plate counts

Control

CFU/ml

O3

Time [min]

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Escherichia coli were also analysed using flow cytometry as outlined above. The data illustrates

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a significant reduction of 85% (ANOVA P value 0.01) in the number of live cells within 16

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minutes of treatment. However, some cells still remained in the lower right (LR) quadrant of the

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graph after treatment. The appearance of live bacterial cells is due to fragments or parts of cell

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walls that can uptake fluorescent stains specific for live cells (SYTO9) and so appear in live cell

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position.


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Figure 6 Effect of ozone on Escherichia coli treated with the USO3 system for 16 minutes

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analysed using flow cytometry

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Control

16 min

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Combined effect of ultrasound and ozone on the viability of Escherichia coli treated using the USO 3 system

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A single bacterial suspension of Escherichia coli was simultaneously treated with ultrasound and

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ozone for 16 minutes. Results illustrate a significant inactivation effect of 99% occurred after 4

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minutes treatment (Figure 7).


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Figure 7 Effect of ultrasound and ozone on the viability of Escherichia coli using the USO3

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system for 16 minutes analysed using viable plate counts Control

CFU/ml

USO3

Time [min]

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Complete inactivation was achieved within 4 minutes, which can be ascribed to a synergistic effect of ultrasound and ozone.

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Flow cytometry data showed a significant reduction of 99% (ANOVA P value 0.01) in the

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number of live cells along with an increase in number of dead cells over 16 minutes treatment

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with ultrasound and ozone (Figure 8). However, a small number of cells (0.6%) appeared to

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remain in the lower right quadrant following treatment.

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Figure 8 Effect of ultrasound and ozone on the viability of Escherichia coli using the USO3

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system for 16 minutes analysed using FCM

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Control

16 min


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Discussion Effect of ultrasound alone on Escherichia coli treated with the USO3 system for 16 minutes

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Bacterial cells treated with ultrasound alone using the USO3 system demonstrated a small

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inactivation effect 24% according to VPC and 26% according to FCM (ANOVA P value 0.05)

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after 16 minutes treatment. This is in accord with previous findings at 512 kHz in that higher

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frequencies at low power have low inactivation effects [26].FCM illustrated a good correlation

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with VPC results supporting this evidence of poor inactivation rates [30] [32].

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Effect of ozone alone on Escherichia coli treated with the USO3system for 16 minutes

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Ozone treatment clearly demonstrated a significant reduction (93% according to VPC and 85%

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according to FCM) (ANOVA P value 0.01) in bacterial cells following 16 minutes treatment.

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Flow cytometry data showed a significant reduction in the number of live cells along with

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increases in the number of dead cells. A study by Sharma reported a promising technique to

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oxidize bacteria in the food and water industry, resulting in more than a 3.5 log reduction in

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viable cells after treatment with 1 ppm ozone for 60 minutes using a selection of bacterial strains

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including the spore forming bacteria Bacillus subtilis [31].

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Effect of combined treatment ultrasound and ozone on Escherichia coli treated with the USO3system for 16 minutes Ultrasound can enhance the effect of ozone on bacteria through a number of processes: 

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De-clumping of bacterial clusters to disperse bacteria as single cells which are more susceptible to oxidation by ozone



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Formation and collapse of cavitation bubbles leads to weakening of the bacterial cells due to breaking chemical bonds in the cell membrane

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

Sonication enhances ozone decomposition rates in water during treatment

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

Sonication increases the level of free radicals and hydrogen peroxide (H2O2) in the

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medium which has bactericidal effects on various microorganisms [25]. Combined

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treatment using the USO3 system produced complete inactivation (99% according to

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VPC and 99% according to FCM) in bacterial cells within 4 minutes treatment.

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Ultrasound clearly enhanced the effect of ozone on bacterial inactivation (ANOVA P

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value 0.01).

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It is clear that the application of (USO3) could significantly contribute in water treatment

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plants. The combination of ozone and ultrasound provides synergetic effects which can

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substantially reduce the cost of treatment and improves health and safety issues associated with

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ozone production. In addition, by employing combined techniques (USO3) can rapidly achieve a

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reliable and safe quality of water for consumers.

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(TEM) evidence of the effect of ultrasound alone for 16 minutes on the biological structure of

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Escherichia coli treated with the USO3system

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TEM imaging was used to show any structural changes on the cells when a Escherichia coli

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bacterial suspension was treated with ultrasound alone. Results showed cell shrinkage and cracks


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in some cell walls and in some cases destruction of the entire cell. Overall approximately 24% of

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bacterial cells had an irregular shape after treatment. An example of a normal and damaged cell

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is shown in (Figure 9).

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Figure 9 (TEM) images showing the effect of ultrasound on the biological structure of

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Escherichia coli treated with the USO3 system (A) control (untreated 24 hour culture, bacterial

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cell with intact cell wall) (B) 16 minutes ultrasound treatment (bacterial cell wall with irregular

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surface and holes due to sonication treatment)

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(A) Control

(B) 16 min treatment

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(TEM) evidence of the effect of ozone alone (1mg/L) on the biological structure of Escherichia coli treated with the USO3 system

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Escherichia coli bacterial suspension was treated with ozone alone to determine the effect of

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ozone on the biological structure of bacterial cells. Results show that ozone produces holes in the

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cell walls as a result of oxidation effects. Overall approximately 86% of bacterial cells had such

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holes in their cell wall and some of the cells had lost their entire cell wall after treatment (Figure

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10).These results are in accord with those of Sharma who demonstrated bactericidal effects of


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ozone result from the disruption of envelope integrity through peroxidation of phospholipids and

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lipoproteins [31].Overall approximately 86% of bacterial cells lost their cell wall after treatment

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(Figure 10).

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Figure 10 (TEM) images showing the effect of ozone alone on the biological structure of

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Escherichia coli treated with the USO3 system (A) control (B) 16 minutes ozone treatment

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(A) Control


363 364 365

Transmission Electron Microscopy (TEM) evidence of the effect of combined treatments of

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ultrasound and ozone on the biological structure of Escherichia coli treated with the USO3

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system. Escherichia coli were subjected to combined treatment with ultrasound and ozone for 16

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minutes using the USO3system. Results illustrate that combined treatment with ultrasound and

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ozone had a significantly greater inactivation effect than separate treatment. TEM images show

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that 95% of the bacteria lost the entire cell walls after treatment, which may be due to the

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synergistic effect of ultrasound and ozone over 16 minutes treatment (Figure 11).

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Figure 11 (TEM) images showing the effect of ultrasound and ozone on the biological structure

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of Escherichia coli treated with the USO3 system (A) control (B) 16 minutes treatment with

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ultrasound and ozone

377 378

(A) Control


379 380 381 382

From the above TEM images it is clear that ultrasound combined with ozone had a great

383

impact on the biological structure of Escherichia coli bacteria. Ozone alone has a significant

384

effect on the bacterial cells but ultrasound enhanced this effect resulting in cell shrinkage due to

385

cell wall removal and the subsequent release of cell contents. This may be due to the combined

386

ultrasonic effects (hydro-mechanical pressures resulting in shear forces and liquid jets which lyse

387

cells, high temperatures and chemical effects due to the production of highly oxidative species of

388

OH● and H2O2), which have a strong anti-microbial action on cells [26].Summary of the (TEM)

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results for bacterial inactivation. The percentage inactivation by TEM was determined using the

390

protocol outlined in the material and methods section and the results are shown (Table 1).

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Table 1 Percentage inactivation in E. coli bacteria 1011 cell/ml treated using the USO3 system for

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16 minutes analysed by TEM

395 Bacterial Ultrasound Strain (612 kHz) E. coli E. coli YES E. coli YES

Ozone (0.5 mg/l) YES YES

Treatment time(min) 16 16 16

% inactivation 86 24 95

396 397 398

Acknowledgment

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The authors would like to thank the Iraqi Ministry of Higher Education who supported this

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research, Dr. Afthab Hussain at Coventry University for assistance with flow cytometry and Mr.

401

Ian Portman director of the imaging suite at Warwick University for support in TEM data

402

analysis.

403 404

References 1. Von Gunten, U. ‘Ozonation of Drinking Water: Part II. ‘Disinfection and by-Product

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Formation in Presence of Bromide, Iodide or Chlorine'. Water Research. 2009, 37 (7), 1469-

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1487

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2. Pascual, A.;Liorca I.;Canut, A. ‘Use of Ozone in Food Industries for Reducing the

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Environmental Impact of Cleaning and Disinfection Activities'. Food Sciences and Technology.

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2009, 18, S29-S35.

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3. Joret, J.; Mennecart, V.; Robert, C.; Compagnon, B.; Cervantes, P. ‘Inactivation of

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Indigenous Bacteria in Water by Ozone and Chlorine'. Water Science and Technology. 1975, 35

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(11-12), 81-86.


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Combined Effect of Ultrasound and Ozone on Bacteria in Water

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