Ozone inactivation microorganisims
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Ozone Inactivation of Microorganisms:Kinetics and Mechanisms
Ahmed Yousef
Professor of Food MicrobiologyOhio State University
Ozone-V ConferenceApril 2, 2007
Fresno California
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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
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Ozone Formation and Decomposition in the Stratosphere(Chapman Mechanism)
vUV
<24
0 nm
v
UV 2
40-3
20 n
m
Atmospheric oxygenmolecules
Atomic oxygenOzone
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Generation of Ozonefor Food Applications
Method• Corona discharge• Electrochemical• Ultraviolet radiation
Consumables• Air• Oxygen gas• Water
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Heat Removal
Heat Removal
AC PowerSupply
Oxygen Ozone
ElectrodeDielectric
High Voltage
Discharge Gap
Ozone Generation by Corona Discharge
Electrode
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H2O
O2/O3
H2
H2O
Anode Cathode
Proton exchange membrane
H+
http://www.lynntech.com/pdf/1lbgenerator.pdf
Ozone Generation by Electrochemical Process
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Ozone Decomposition and Disposal
- Heat
• Destruction of excess ozone in work environment
• Destruct units:
- Catalysts
• Small amounts
- May dispose of in the atmosphere
For ozone factsheet, visit {http://ohioline.osu.edu/fse-fact/0005.html}
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Inactivation Kinetics
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Inactivation of food-transmitted microorganisms (vegetative cell in pure suspensions) by aqueous ozone
(Kim & Yousef, 2000)
Ozone kills diverse bacteria
Spoilage and pathogenic bacteria are inactivated
Rapid inactivation
Ozone kills bacteria in less than 30s
Effective at low concentrations
~1ppm ozone kills up to 6 logs 0 200 400Exposure time (sec)
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
Frac
tion
of S
urvi
vors
(N/N
o)
1.44 ppm
0.96 ppm
1.52 ppm
1.12 ppm
Escherichia coli O157:H7
Pseudomonas fluorescens
Leuconostoc mesenteroides
Listeria monocytogenes
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0.00 0.01 0.02 0.03Ozone dose (mg/ml)
1.0E-7
1.0E-6
1.0E-5
1.0E-4
1.0E-3
1.0E-2
1.0E-1
1.0E+0A. acidocaldarius (cell)
A. acidocaldarius (spore)
N. fischeri (spore)
Z. bailli (spore)
Inactivation of bacterial and fungal spores suspended in water by ozoneInitial count: 6.4x106 -1.5x107 cfu/ml (Khadre et al., 2001)
Surv
ivor
frac
tion
(N/N
0)
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An ozone dose (mg gas ozone/mL sample) =Ozone concentration in gas (mg/L) × flow rate (mL/min) ×treatment time (min)/volume of spore suspension (mL).
(We apologize for the inconvenience)
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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)
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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 22°C(Khadre & Yousef, 2001)
1.35.7B. subtilis vary Niger ATCC 9372
0.646.1B. subtilis ATCC 19659
1.24.8B. subtilis OSU848
0.322.7B. subtilis OSU494
0.641.3B. stearothermophilus OSU24
0.581.9B. polymyxa OSU443
0.932.1B. megaterium OSU125
1.66.1B. cereus OSU11H2O2O3Spore
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Scanning electron micrograph of rotavirus particlesafter release from MA 104 cell culture
Khadre and Yousef, 2002
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0 5 10 15 20 25 30Ozone Concentration (ppm)
2
4
6
8
10
12
Log 1
0 TC
ID50
/mL
Changes in infectivity of rotavirus Wa Wooster, measured as TCID50/mLat different concentrations of ozone in aqueous solution at 25°C.
Trial 1
Trial 2
Khadre and Yousef, 2002
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What do these kinetic data mean?
- Cell suspension (planktonic) vs. biofilm- Equipment vs. package surface- Medium more complicated than pure water
• Lab research vs. Real World• Testing different scenarios
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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 exposureto1-min treatments with ~0.1 mg ozone/chip using 3.6 ppm aqueous ozone (Khadre & Yousef, 2000).___________________________________________________________No. of Exposures Biofilm Dry film______________________________________________________0 3.5x108 7.2x108
1 3.2x106 6.4x103
2 2.7x105 <1(est)3 2.2x105
4 1.2x105
5 6.0x102
______________________________________________________
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0 4 8 12 16Ozone Concentration (PPM)
2.0
4.0
6.0
8.0
Log
CFU
/Chi
p
0.00 0.08 0.16 0.24 0.32
mg Ozone/Chip
24
StainlessSteel
Packaging Material
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)
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Ozone lethality against Escherichia coli O157:H7 in the presence of organic load (BSA). Restaino et al., 1995; Achen, 2000
2
3
4
5
6
7
8
9Lo
g cf
u/m
l
0 0.5 1.2 1.8 3.5Ozone (ppm)
Control
0.01% BSA
0.1% BSA
1% BSA
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Inactivation Mechanism
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Oxidation Potential of Selected Oxidizing Agents
0.700.95Chlorine dioxide
0.981.33Hypobromite
1.001.36Free chlorine
1.091.48Hypochlorite
1.311.78Hydrogen peroxide
1.532.08Ozone
Relative Oxidative
Powera
Oxidation Potential
(Volts)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.
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.OH
O3Initiators
OH-, Fe2+, UV, H2O2(Radicals formed)
HO2. .O2
-
O3
O2
PromotorsO3, -SH, R-CH2OH, Aryl
(.O2- regenerated, O3 consumed)
InhibitorsAlkyl, t-BuOH, CO3
2+/HCO3+
Radicals Consumed(Ozone decomposition terminated)
Ozone decomposition, free radical formationand advanced oxidation processes
(Khadre et al, 2001)
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Oxidative powerMolecular ozone (Hunt & Marinas, 1997)Singlet, free radicals (Kanofsky & Sima, 1991)
Inactivation Mechanism
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Reaction with:Cell membranes (Giese & Christenser, 1954)Dehydrogenases (Ingram & Haines, 1955)DNA (Scott, 1975)RNA (Kin et al., 1980)
Inactivation Mechanism (Cont’d)
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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.
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• Inner membrane damage is the probable killing mechanism for ozone(Young, 2004)
• Oxidizing agents may have targeted proteins, not lipids, in the spore’s inner membrane(Cortezzo et al., 2004).
Target in sporeInner membrane!
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Future Directions
Combination Treatments(if justifiable)
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D-values* (min) of spores treated with ozoneTemperature (°C) Treatment
85 90 95 Control (no ozone)
294.1 74.6 27.0
Ozone-treated (before heating)
26.3 9.3 4.0
Kim et al., 2002* The smaller the D-value, the greater the sensitivity to heat
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• 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 pre-treated with sublethal levels of ozone.
• Direct use of ozone in liquid foods and on food surfaces with large ozone demand may not be recommended.
Conclusions