Appl Microbiol Biotechnol (1998) 49: 766±769
ORIGINAL PAPER
I. R. Komanapalli á B. H. S. Lau
Inactivation of bacteriophage k, Escherichia coli, and
Candida albicans by ozone
Received: 15 October 1997 / Accepted: 24 February 1998
Abstract The e€ects of ozone (O3) on three types of mi-
crobes were studied. Test suspensions were exposed to
600 ppm O3 at room temperature. Control experiments
were performed under identical conditions using oxygen
gas. Bacteriophage k was completely inactivated at
10 min while Escherichia coli and Candida albicans were
only inactivated by factors of 105 and 104 respectively at
40 min. Exposure of a mixed microbial suspension to O3
for 5 min resulted in 100% killing of bacteriophages while
the viability of E. coli remained unchanged. Various body
¯uids containing phages were exposed to O3. Compared
to bu€ered solution, the decrease in phage titers was
signi®cantly slower in whole blood, plasma, and albumin.
Both E. coli and C. albicans had increased production of
thiobarbituric-acid-reactive substances with increased O3
exposure. 3H-labelled amino acids were incorporated into
E. coli. O3 treatment resulted in a loss of radioactivity,
indicating leakage of cytoplasmic contents. The data in-
dicate that microbes are inactivated by O3 at di€erent
rates, possibly related to di€erential membrane perme-
ability. The milieu in which microbes are present deter-
mines the e€ectiveness and outcome of O3 treatment.
Introduction
Ozone is widely used for water treatment in Europe and
is now receiving considerable attention in the United
States and several other countries. The e€ects of ozone
on various microorganisms have received much atten-
tion because of its increasing use in water and sewage
disinfection (Katzenelson and Biedermann 1976; Boyce
et al. 1981; Glaze 1987). Inactivation kinetics with ozone
has been studied on the laboratory and pilot-plant scale
I. R. Komanapalli á B. H. S. Lau (&)
Department of Microbiology and Molecular Genetics,
School of Medicine, Loma Linda University,
Loma Linda, California 92350, USA
Tel.: +1 909 824 4480
Fax: +1 909 824 4035
e-mail: bLau@ccmail.llu.edu
Ó Springer-Verlag 1998
using a variety of microorganisms (Sommerville and
Rempel 1972; Keller et al. 1974). Damage to the coat
protein is thought to be responsible for inactivation of
bacteriophage /X174, f2, and poliovirus type 2 (De Mik
and De Groot 1977; Reisser et al. 1977; Kim et al. 1980).
The inactivation of bacteria and fungi is more complex,
since ozone attacks protein and unsaturated lipids of
both cell membrane and cytoplasmic components (Scott
and Lesher 1963; Mudd et al. 1969; Hinze et al. 1987;
Pryor et al. 1991). Ozone has been reported to be useful
in decontamination of bioclean rooms (Masaoka et al.
1982). Ozonization of autologous blood followed by
slow reinfusion into patients, has been used for a long
time in Western Europe for the treatment of various
viral diseases (Rilling 1985). The therapeutic bene®ts
observed in the autohemotherapy procedure are thought
to be due to the activation of the immune system (Bocci
and Paulesu 1990).
Our recent studies with Escherichia coli indicate that
membrane components are primary targets of ozone
damage with subsequent reactions involving the intra-
cellular components, protein and DNA (Komanapalli
and Lau 1996). We further showed that the sulfhydryl
group in the membrane is most susceptible to ozone
inactivation (Komanapalli et al. 1997). In this study, the
e€ects of ozone on three types of microbes: bacterio-
phage k, E. coli, and Candida albicans, were determined.
We also studied the e€ect of ozonation of body ¯uids
contaminated with bacteriophage k. Oxidative damage
of the membrane after ozone exposure was measured by
production of thiobarbituric-acid-reactive substances
from C. albicans and E. coli and leakage of a labelled
amino acid mixture from E. coli.
Materials and methods
Materials
E. coli K-12 (CSH50, Cold Spring Harbor Laboratory) and bac-
teriophage k were obtained from Dr. Jun-Ichi Ryu of Loma Linda
 767

University, Loma Linda, California. Human albumin was obtained
from Immuno-U.S. Inc., (Rochester, Mich.). Human whole blood
and plasma and C. albicans were from the clinical laboratory of
Loma Linda University Medical Center, Loma Linda, Calif. Ozone
was generated from pure oxygen by an ozonizer (Ultraviolet
Products Inc., San Gabriel, Calif.). The concentration of ozone
output was determined spectrophotometrically using the potassium
iodide method (Shechter 1973). Test suspensions (5 ml containing
108 organisms) were exposed to 107 nmol O3/min (600 ppm), at a
constant ¯ow rate of 20 ml/min for various periods by permitting
the gas to bubble directly into the suspensions at room temperature
(22 °C). Control experiments were performed under identical
conditions using oxygen gas.
and counted in a Beckman model LS3801 scintillation counter
(Beckman Instruments Inc., Fullerton, Calif.) to determine the
amount of 3H-labelled amino acid mixture retained inside the cells.
Statistical analyses
Data were analyzed with one-way analysis of variance followed by
Tukey's multiple-range test for signi®cant di€erence. Results were
expressed as the mean ‹ SE. A P value less than 0.05 was con-
sidered signi®cant. All statistical procedures were performed with
Statgraphics software version 5.0 (STSC Inc., Rockville, Md.).

Media Results

Luria-Bertani (LB) broth consisted of 10 g Bacto Tryptone (Difco,
Detroit, Mich.), 5 g yeast extract (Difco), 5 g NaCl and 1 g dex-
trose/l. In addition, 15 g (1.5% w/v) of Bacto Agar (Difco) was
added for LB agar plates. For phage titration, 10 g (1% w/v) Bacto
agar (Difco) was used instead. LB broth with maltose (0.4% w/v)
substituted for dextrose was used for plating phage k. Sabouraud
dextrose/agar (Difco) was used for C. albicans.
Preparation and titration of bacteriophage k
Bacteriophage k was propagated on the appropriate host (E. coli K-
12) by a modi®ed plate method (Davis et al. 1980). After treatment
with ozone, the phage suspensions were serially diluted and the
plaque-forming units per milliliter (pfu/ml) were determined.
Growth condition and determination of cell viability
E. coli K-12 and C. albicans were used for comparison of cell sur-
vival and lipid oxidation. Aliquots of untreated and treated samples
were removed and appropriate dilutions plated on LB medium for
E. coli and Sabouraud dextrose/agar for C. albicans. The plates
were incubated overnight at 37 °C and the colonies counted and
expressed as colony-forming units per milliliter (cfu/ml).
Determination of lipid oxidation
The treated and untreated suspensions were centrifuged at
2000 rpm for 10 min at 22 °C and supernatants collected for de-
termination of thiobarbituric-acid-reactive substances by the ¯uo-
rometric assay (Yagi 1976). Fluorometric measurements were made
at 553 nm with 515 nm excitation, using a LS-3 ¯uorescence
spectrophotometer (Perkin-Elmer, Norwalk, Conn.). The ¯uores-
cence value was calculated by comparing with standards prepared
from tetraethoxypropane (Sigma Chemical Co. St. louis, Mo.).
Oxygen gas had no e€ect on E. coli while ozone treat-
ment resulted in a time-dependent decrease of cell via-
bility (Fig. 1). Bacteriophage k was extremely sensitive
to ozone with a 104-fold inactivation at 8 min and in-
activation by more than a factor of 109 at 10 min
(Fig. 2). In contrast, both E. coli and C. albicans showed
a 100-fold decrease at 10 min. At 40 min of ozone ex-
posure, the viability decreased by a factor of 105 in E. coli
and 104 in C. albicans. Study of the comparative re-
sponse of mixed cultures of bacteriophages and bacteria
shows that k titers dropped signi®cantly within 5 min of
ozone exposure while E. coli viability remained un-
changed during this interval (Fig. 3). Bacteriophage
k was 100% inactivated at 10 min in the phosphate
bu€er solution. In di€erent body ¯uids (10% plasma,
5% albumin, and whole blood), complete inactivation of
k was delayed until 25±30 min exposure (Fig. 4). During
this time the ¯uids were also oxidized, as indicated by the
disappearance of color or the so-called bleaching e€ect.
Ozone-induced lipid oxidation in E. coli and C. albi-
cans is shown in Fig. 5. A dose-dependent increase in the
production of thiobarbituric-acid-reactive substances
was seen with both organisms. The amount of thiobar-
bituric-acid-reactive substances produced by C. albicans
was twice as much as that by E. coli at 40 min. Figure 6

Incorporation of 3H-labelled amino acid mixture

E. coli cells, harvested at mid-log phase, were washed and resus-

pended in phosphate bu€er, pH 7.0, to an absorbance reading of
0.8 at 520 nm (1 ´ 108 cells/ml). Chloramphenicol (200 lg/ml) was
added to the cell suspension and incubated at 35 °C for 10 min
with shaking to inhibit protein synthesis. 3H-labelled amino acid
mixture (5 lCi/ml, ICN Pharmaceuticals, Costa Mesa, Calif.) was
then added and, 2 min later, cells were washed and resuspended in
phosphate bu€er. The cell suspensions (5 ml) were then treated
with ozone for di€erent periods (0, 1, 2, 3, 4, and 5 min). Samples
exposed to oxygen were used as controls. Immediately after each
exposure, the cell suspension was pipetted onto Millipore HA ®lters
(25 mm diameter, 0.45 lm pore size, Millipore Corp., Bedford,
Mass.) on a Millipore vaccum manifold. The cells retained on the Fig. 1 Comparison of cell viability of Escherichia coli K-12 on ozone
®lter were rapidly rinsed four times with 1-ml aliquots of 0.85% (600 ppm) and oxygen exposure. Bars represent means ‹ standard
saline. The washed ®lters were placed in 10 ml CytoScint cocktail errors of triplicate samples. Asterisk signi®cant di€erence (P < 0.05)
(ICN Biomedicals, Irvine, Calif.) in 20-ml glass scintillation vials from respective control at 0 point