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Ozone Inactivation of Microorganisms: Kinetics and Mechanisms Ahmed Yousef Professor of Food Microbiology Ohio State University Ozone-V Conference April 2, 2007 Fresno California


What’s Ozone?

• • • • • •

Tri-atomic oxygen (O3) Molecular weight of 48 Bluish gas (at high concentrations) Pungent characteristic odor Low solubility in water Half-life: • Gas: ~12 hr (at ambient) • Aqueous: Short, varies by medium


40 -32 0n

m

UV

<2 40 nm

Ozone Formation and Decomposition in the Stratosphere (Chapman Mechanism)

Atmospheric oxygen molecules

UV 2

v

v Atomic oxygen Ozone


Generation of Ozone for Food Applications Method • Corona discharge • Electrochemical • Ultraviolet radiation

Consumables • Air • Oxygen gas • Water


Ozone Generation by Corona Discharge Heat Removal Electrode Dielectric

High Voltage

AC Power Supply

Oxygen

Discharge Gap

Ozone Electrode

Heat Removal


Ozone Generation by Electrochemical Process

Anode

H 2O

O2/O3

Cathode

Proton exchange membrane

H2

H+

H 2O

http://www.lynntech.com/pdf/1lbgenerator.pdf


Ozone Decomposition and Disposal • Destruction of excess ozone in work environment • Destruct units: - Heat - Catalysts • Small amounts - May dispose of in the atmosphere For ozone factsheet, visit {http://ohioline.osu.edu/fse-fact/0005.html}


Inactivation Kinetics


1

Spoilage and pathogenic bacteria are inactivated

Rapid inactivation Ozone kills bacteria in less than 30s

Fraction of Survivors (N/No)

Ozone kills diverse bacteria

Escherichia coli O157:H7

0.1

0.96 ppm 0.01

1.52 ppm Leuconostoc mesenteroides

0.001

Pseudomonas fluorescens 1.44 ppm

0.0001

0.00001

Listeria monocytogenes

1.12 ppm

Effective at low concentrations ~1ppm ozone kills up to 6 logs

0.000001 0

200

Exposure time (sec)

Inactivation of food-transmitted microorganisms (vegetative cell in pure suspensions) by aqueous ozone (Kim & Yousef, 2000)

400


1.0E+0 A. acidocaldarius (cell) A. acidocaldarius (spore)

Survivor fraction (N/N0)

1.0E-1

N. fischeri (spore)

1.0E-2

Z. bailli (spore)

1.0E-3 1.0E-4 1.0E-5 1.0E-6 1.0E-7 0.00

0.01

0.02

0.03

Ozone dose (mg/ml) Inactivation of bacterial and fungal spores suspended in water by ozone Initial count: 6.4x106 -1.5x107 cfu/ml (Khadre et al., 2001)


An ozone dose (mg gas ozone/mL sample) = Ozone concentration in gas (mg/L) Ă&#x2014; flow rate (mL/min) Ă&#x2014; treatment time (min)/volume of spore suspension (mL).

(We apologize for the inconvenience)


Treatment of Clostridium botulinum spores with aqueous ozone for 1 min

Treatment

Viable spores/ mL

Control (0 ppm)

3.6 x 107

12 ppm

< 1 (estimated)

26 ppm

< 1 (estimated)


Decrease in spore count (log10/ml) with exposure to ozone (0.22 mg ozone/20 ml mixture) or hydrogen peroxide (2000 mg H2O2/20 ml mixture) for 1 min at 22째C (Khadre & Yousef, 2001)

Spore

O3

H2O2

B. cereus OSU11

6.1

1.6

B. megaterium OSU125

2.1

0.93

B. polymyxa OSU443

1.9

0.58

B. stearothermophilus OSU24

1.3

0.64

B. subtilis OSU494

2.7

0.32

B. subtilis OSU848

4.8

1.2

B. subtilis ATCC 19659

6.1

0.64

B. subtilis vary Niger ATCC 9372

5.7

1.3


Scanning electron micrograph of rotavirus particles after release from MA 104 cell culture Khadre and Yousef, 2002


12

Log10 TCID50 /mL

10 Trial 1

8

6 Trial 2

4

2

0

5

10

15

20

25

30

Ozone Concentration (ppm)

Changes in infectivity of rotavirus Wa Wooster, measured as TCID50/mL at different concentrations of ozone in aqueous solution at 25째C. Khadre and Yousef, 2002


What do these kinetic data mean? â&#x20AC;˘ Lab research vs. Real World â&#x20AC;˘ Testing different scenarios - Cell suspension (planktonic) vs. biofilm - Equipment vs. package surface - Medium more complicated than pure water


Efficacy Against Biofilm-Repeated Exposure Count of Pseudomonas fluorescens as a biofilm or a dry film on chips (12.9 cm2) of a multilaminated packaging material after repeated exposure to1-min treatments with ~0.1 mg ozone/chip using 3.6 ppm aqueous ozone (Khadre & Yousef, 2000). ___________________________________________________________

No. of Exposures Biofilm Dry film ______________________________________________________ 7.2x108 0 3.5x108 1 3.2x106 6.4x103 2 2.7x105 <1(est) 3 2.2x105 4 1.2x105 5 6.0x102 ______________________________________________________


Log CFU/Chip

8.0

Packaging Material

6.0

4.0

Stainless Steel

2.0

0

4

8

12

16

Ozone Concentration (PPM) 0.00

0.08

0.16

mg Ozone/Chip

0.24

0.32

Inactivation of 24-hr biofilm of Pseudomonas fluorescence on chips (12.9-cm2) of packaging material and stainless steel when exposed to different doses of ozone (Khadre & Yousef, 2000) 24


9 1% BSA

Log cfu/ml

8

0.1% BSA

7 6

0.01% BSA

5 4 Control

3 2

0

0.5

1.2 Ozone (ppm)

1.8

3.5

Ozone lethality against Escherichia coli O157:H7 in the presence of organic load (BSA). Restaino et al., 1995; Achen, 2000


Inactivation Mechanism


Oxidation Potential of Selected Oxidizing Agents Oxidation Potential (Volts)

Relative Oxidative Powera

Ozone

2.08

1.53

Hydrogen peroxide

1.78

1.31

Hypochlorite

1.48

1.09

Free chlorine

1.36

1.00

Hypobromite

1.33

0.98

Chlorine dioxide

0.95

0.70

Species

a

relevant to chlorine

Water Quality Association Ozone Task Force. 1997. Ozone for Point-of-Use, Point-of-Entry, and Small System Water Treatment Applications: A Reference Manual.Water Quality Association. Lisle, IL, 2-4.


O3 Initiators OH-,

Fe2+, UV, H2O2 (Radicals formed)

.O -

.

HO2

2

Promotors (.O2-

O3, -SH, R-CH2OH, Aryl regenerated, O3 consumed)

.OH

O3 O2

Inhibitors Alkyl, t-BuOH, CO32+/HCO3+

Radicals Consumed (Ozone decomposition terminated)

Ozone decomposition, free radical formation and advanced oxidation processes (Khadre et al, 2001)


Inactivation Mechanism Oxidative power Molecular ozone Singlet, free radicals

(Hunt & Marinas, 1997) (Kanofsky & Sima, 1991)


Inactivation Mechanism (Contâ&#x20AC;&#x2122;d) Reaction with: Cell membranes Dehydrogenases DNA RNA

(Giese & Christenser, 1954) (Ingram & Haines, 1955) (Scott, 1975) (Kin et al., 1980)


Ozone action on bacterial spores Ozone at 5 ppm Damages spores coats (see the electron microscopic pictures). Ozone at >5ppm Total inactivation of spores (data not shown)

Before

After

Khadre, M. A. and Yousef, A.E. 2001. Sporicidal action of ozone and hydrogen peroxide, a comparative study. Int. J. Food Microbiol. 71:131-138.


Target in spore Inner membrane! â&#x20AC;˘ Inner membrane damage is the probable killing mechanism for ozone (Young, 2004) â&#x20AC;˘ Oxidizing agents may have targeted proteins, not lipids, in the sporeâ&#x20AC;&#x2122;s inner membrane (Cortezzo et al., 2004).


Future Directions

Combination Treatments (if justifiable)


D-values* (min) of spores treated with ozone Treatm ent

Control (no ozone) Ozone-treated (before heating)

Tem perature (째C) 85

90

95

294.1

74.6

27.0

26.3

9.3

4.0

Kim et al., 2002 * The smaller the D-value, the greater the sensitivity to heat


Conclusions • Ozone inactivates microbial cells rapidly and effectively. • Spores of Bacillus and Clostridium species, compared to vegetative cells, require higher ozone concentrations to be killed. • Ozone damages spore outer coats but membrane damage is probably the cause ozone sporicidal action. • Bacterial spores become sensitive to heat when pretreated with sublethal levels of ozone. • Direct use of ozone in liquid foods and on food surfaces with large ozone demand may not be recommended.

Ozone inactivation microorganisims  

Ahmed Yousef Professor of Food Microbiology Ohio State University Ozone-V Conference April 2, 2007 Fresno California • Tri-atomic oxygen (O...

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