INACTIVATION OF ESCHERICHIA COLI WITH OZONE:
CHEMICAL AND INACTIVATION KINETICS
NIMRATA K. HUNT
1
and BENITO J. MARINÄAS
2
*
*
M
1
Woodard and Curran Environmental Services, Portland, ME 04102, USA;
2
Department of Civil and
Environmental Engineering, University of Illinois, Urbana, IL 61801, USA
(First received August 1998; accepted in revised form December 1998)
AbstractÐThe apparent chemical and inactivation reactions taking place during the disinfection of
Escherichia coli with ozone in the presence of humic acid were investigated with continuous-¯ow tubu-
lar reactors. The apparent decomposition of dissolved ozone in the presence of humic acid and E. coli
cells was modeled successfully with mixed second-order rate expressions within a time scale relevant to
E. coli inactivation with ozone. The rate for the ozone inactivation of E. coli in the presence of humic
acid was slower than that in the absence of natural organic matter due to the faster decomposition of
dissolved ozone and thus the lower exposure of E. coli cells to the disinfectant in the former case. How-
ever, the second-order inactivation rate constant was approximately the same in the presence and
absence of humic acid con®rming that molecular ozone rather than radicals was the species generally
responsible for inactivation. The overall rate of reaction between ozone and organic matter associated
with E. coli cells was slower that the rate of E. coli inactivation by ozone. Only 25% of the initial rela-
tively fast ozone demand were satis®ed by the time that practically all micro-organisms present in sol-
ution were inactivated. TEM micrographs revealed that noticeable changes in the interior of E. coli
cells did not take place until most of the cells present in the sample were non-viable. # 1999 Elsevier
Science Ltd. All rights reserved
Key wordsÐcontinuous-¯ow reactor, Escherichia coli, humic acid, inactivation kinetics, ozone disinfec-
tion, transmission electron microscopy
INTRODUCTION
The disinfection of Escherichia coli (E. coli) with
ozone has been investigated with several con®gur-
ations of semi-batch and continuous-¯ow through
reactors (Perrich et al., 1975; Zhou and Smith,
1994; Hunt and MarinÄas, 1997). In general, the in-
activation kinetics were found to be pH indepen-
dent and consistent with the classic Chick±Watson
model (Chick, 1908; Watson, 1908) according to the
batch reactor expression:
dN
dt
ÿk
i
cN,
1
where N is the density of viable micro-organisms, c
is the concentration of dissolved molecular ozone,
both at time t from the start of the reaction, and k
i
is the inactivation rate constant given by (Hunt and
MarinÄas, 1997):
k
i
A exp
ÿ
E
a
RT
2
in which E
a
=37,100 J/mol is the activation energy,
A=5.3710
8
l/(mg s) is the frequency factor,
R=8.314 J/(mol K) is the ideal gas constant and T
is absolute temperature in K. Deviations from
equation 1 including inactivation curve shoulders
and tails have been attributed to departure of ex-
perimental units from ideal reactor behavior and
the occurrence of small fractions of bacterial clumps
(Hunt and MarinÄas, 1997).
The consistency of experimental data with
equation 1 indicates that the inactivation kinetics
could be modeled with a homogeneous second
order rate law. However, the inactivation process is
a more complex heterogeneous phenomenon includ-
ing various mass transfer steps and reactions. The
disinfectant must diuse toward the surface of the
micro-organism, and then permeate into the mem-
brane and cytoplasm. The rate of mass transfer
could be aected by various intracellular and extra-
cellular reactions with biomolecules. Inactivation or
loss of viability would occur when vital constituents
would suer a certain level of irreversible damage.
An important concept is that the chemical reactions
between ozone and biomolecules continue for a
relatively longer period of time after loss of viability
until the disinfectant is depleted or the oxidation
demand by biomolecules is satis®ed.
Wat. Res. Vol. 33, No. 11, pp. 2633±2641, 1999
# 1999 Elsevier Science Ltd All rights reserved
Printed in Great Britain
0043-1354/99/$ - see front matter
PII: S0043-1354(99)00115-3
*Author to whom correspondence should be addressed.
[Tel.: +1-217-3336961; fax: +1-217-3339464; e-mail:
marinas@uiuc.edu].
2633
Despite decades of research eorts by various
groups, the mechanisms responsible for the inacti-
vation of E. coli are still not well understood.
Christensen and Giese (1954) suggested that the
bacterial membrane was the ®rst site of ozone
attack. In agreement with this suggestion, Scott and
Lesher (1963) indicated that ozone attacked the cell
surface, altering the permeability of the membrane
and ultimately resulting in leakage of cell contents
into the medium. The occurrence of leakage was
con®rmed by showing that the concentrations of
nucleic acids and proteins increased in the medium,
and decreased inside cells. Though ozone aected
the aggregation of nucleoproteins inside the cells, it
did not react appreciably with the nucleic acids
prior to being released into the medium. Further
evidence of the inability of ozone to penetrate the
cells was given by the fact that the highly reactive
reduced sulfur groups of glutathione inside the cells
were unaected, while in contrast ozone reacted
very fast with glutathione present in the medium.
More recently, other authors have reported that
proteins and unsaturated lipids in the cell mem-
brane are the targets of ozone attack (Pryor et al.,
1983) and leakage of cell contents into the medium
and ultimately cell lysis can result after prolonged
ozonation (Scott, 1975; Hamelin et al., 1978).
Hamelin et al. (1978) suggested that ozone pro-
duced single strand breaks in DNA which, if unre-
paired, caused extensive breakdown of DNA in E.
coli ultimately resulting in the loss of viability.
Besides these lesions, ozone might also induce base
damage and protein cross-linking in DNA mol-
ecules (Hamelin et al., 1977).
Perrich et al. (1975) concluded that cell lysis was
not the main mechanism for inactivation of E. coli
and that the cells remained morphologically intact
after inactivation. Ishizaki et al. (1987) determined
that ozone aected plasmid DNA harbored in E.
coli cells by converting the closed circular DNA to
open circular DNA. This observation indicated that
ozone was able to diuse through the cell mem-
brane and react with cell constituents. The authors
proposed that damage to chromosomal DNA might
be one of the reasons for inactivation of E. coli by
ozone. Hamelin and Chung (1974) studied the abil-
ity of ozone to induce mutations in E. coli and pos-
tulated that ozone was able to penetrate into the
cell and genetically alter the cytoplasmic constitu-
ents before destroying the cell membrane. They
determined that inactivation was a function of the
amount of ozone available per bacterium, and
below a certain threshold concentration, ozone had
no eect on the survival of bacteria.
Disinfection of E. coli in natural water and
wastewater presents an additional degree of com-
plexity because ozone will also react with dissolved,
colloidal and particulate matter, and these reactions
might interfere with some of the reactions respon-
sible for E. coli inactivation. Designing disinfection
reactors thus might require the simultaneous con-
sideration of all reactions aecting the concen-
tration of dissolved ozone and ultimately the
inactivation process.
The main objective of this study was to investi-
gate and model the apparent chemical and inacti-
vation reactions taking place during the disinfection
of E. coli with ozone in the presence of humic acid,
a group of compounds selected to represent a frac-
tion of the organic matter present in natural waters.
An additional objective was the assessment of E.
coli cell structure transformation at various levels of
exposure to ozone.
MATERIALS AND METHODS
Experimental design
The scope of work of this study included the perform-
ance of four experimental sets. Two sets were designed to
investigate separately the apparent overall decomposition
of ozone reacting with E. coli and humic acid. The third
experimental set was performed to con®rm that the appar-
ent kinetics determined for the separate reactions with the
previous two sets could be used to represent the overall
decomposition of ozone in the simultaneous presence of E.
coli and humic acid. The objective of the fourth set was to
determine the eect of the presence of humic acid on the
inactivation kinetics of E. coli with ozone. All experiments
were performed with a continuous-¯ow tubular reactor ap-
paratus described previously (Hunt and MarinÄas, 1997). A
summary of experimental conditions used is presented in
Table 1. Experimental set I also included tests performed
with stock solutions of E. coli sonicated in a Fisher 50
Sonic Dismemberator (Fisher Scienti®c, Itasca, IL) to
assess potential mass transfer limitations aecting the
overall inactivation process. Complete cell lysis resulting
from the sonication treatment was con®rmed microscopi-
cally.
Table 1. Summary of initial ozone (c
o
), E. coli (N
o
) and humic acid (TOC
o
) concentrations.
Additional conditions applicable to all experimental sets unless otherwise indicated:
temperature=208C,
pH=6,
total
phosphate
concentration=0.02 M,
tert-butanol
concentration=0.01 M, tubular reactor ¯ow rate=50 ml/min, contact time (t)=3.8±48.4 s
Experimental set
c
o
(mg/l)
N
o
(10
9
CFU/l)
TOC
o
(mg/l)
I
65±266
0.35±7.2
0
II
26±304
0
0±1.0
III$
190±374
7.0±13.
0±0.50
IV
6±52
5.7±13
0±0.50
$
Tubular reactor ¯ow rate=65 ml/min, t=2.5±47.8 s.
Nimrata K. Hunt and Benito J. MarinÄas
2634
Materials
Stock solutions of ozone, E. coli and humic acids were
prepared in ozone demand free water buered at pH 6.0
or 7.2 with 0.02 M phosphate. E. coli ATCC strain 11775
(American Type Culture Collection, Rockville, MD) was
used as the test micro-organism. Humic acid was obtained
from
Aldrich
Chemical
Co.
(Milwaukee,
WI).
Experimental solutions were dosed with 0.01 M tert-buta-
nol for radical scavenging and the temperature of all tests
was maintained constant at 208C by means of immersing
the tubular reactors in a water bath. Additional infor-
mation about experimental, chemical and microbial com-
ponents was presented in a previous publication (Hunt
and MarinÄas, 1997).
Analytical methods
Total organic carbon (TOC) analyses were performed to
determine the organic carbon concentrations correspond-
ing to the various humic acid dosages used as well as to
quantify the organic carbon content of E. coli cells. The
latter analyses were performed by sonicating 10 ml of
stock E. coli solution in a chilled water bath for approxi-
mately 10 min, the time required for complete cell lysis as
con®rmed by microscopic examination. A few drops of
concentrated phosphoric acid (85%) were added to the
sonicated cells prior to sample analyses with a TOC analy-
zer Dohrmann Model DC-80 (Tekmar-Dohrmann,
Cincinnati, OH). Analytical procedures for ozone determi-
nation and E. coli viability assessment were described pre-
viously (Hunt and MarinÄas, 1997).
Transmission electron microscopy
Additional inactivation experiments were performed to
study structural changes of E. coli cells after several levels
of exposure to ozone. Experimental conditions included
initial ozone concentrations c
o
=0±1000 mg/l, E. coli den-
sity N
o
=510
9
CFU/l and contact time t=30 s. The ex-
periments were performed at 208C and pH 7.2 in 0.02 M
phosphate buer solution in the presence of 0.01 M tert-
butanol. For each test, 50-ml sample volumes were col-
lected in bottles containing 10 ml of 0.2% sodium thiosul-
fate solution to quench excess ozone. The samples were
centrifuged to concentrate the micro-organisms, which
were then ®xed with an equal volume of 3% glutaralde-
hyde (Electron Microscopy Sciences, Fort Washington,
PA) buered at pH 7.2 with phosphate. After approxi-
mately 1 h, the samples were centrifuged and the resulting
bacterial pellets were exposed overnight to additional
phosphate-buered 3% glutaraldehyde solution.
The glutaraldehyde-®xed bacteria were embedded in 1%
agar and washed twice in phosphate buer. The samples
were then ®xed in buered 1% osmium tetroxide/1.5%
potassium ferrocyanide at 48C for 1.5 h. The ®xed samples
were rinsed in phosphate buer twice and dried by ex-
posure to gradually increasing concentrations of ethanol
(30, 60, 90, 100 and 100%). The dried samples were rinsed
twice in propylene oxide and then in®ltrated with propy-
lene oxide±epoxy resin Poly/Bed 812 (Polysciences,
Warrington, VA) mixtures until the samples were in pure
epoxy resin. The samples were placed in polyethylene cap-
sules and the resin polymerized at 608C for 48 h. Thin sec-
tions were cut on a Reichert-Jung Ultracut E
ultramicrotome (Carl Zeiss, Thornwood, NY) and stained
with uranyl acetate and lead citrate. The sections were
examined with a JEOL JEM-100CX transmission electron
microscope (TEM) (JEOL USA, Peabody, MA) operating
at 80 kV and micrographs were recorded on Kodak SO-
163 electron image ®lm (Eastman Kodak Co., Rochester,
NY) and subsequently digitized with a high-resolution
scanner.
RESULTS AND DISCUSSION
Experimental results obtained for the tests per-
formed to assess the overall decomposition kinetics
of ozone in the presence of E. coli are presented in
Fig. 1. Modeling eorts revealed that the apparent
ozone decomposition could be represented with a
mixed second order rate expression or
dc
dt
ÿk
x
cx,
3
where k
x
is a second order rate constant in l/(mg s)
and x is the concentration of fast ozone demand
constituents in E. coli cells in mg/l as O
3
given by:
x c ÿ c
o
x
o
4
in which x
o
is the initial concentration of fast
ozone-demand constituents, a quantity proportional
to the initial number of viable micro-organisms N
o
or x
o
=a
o
N
o
. a
o
can be expressed in mg of O
3
per
colony forming unit (mg O
3
/CFU) or the ozone
demand per E. coli cell assuming that each bacterial
colony resulted from an individual cell.
Integration of equation 3 after substitution of
equation 4 resulted in the following expression:
Fig. 1. Apparent overall decomposition of ozone in the
presence of initially viable E. coli for various initial ozone
concentrations and micro-organism densities (see set I in
Table 1 for experimental conditions).
Inactivation of E. coli with ozone
2635
c
c
o
c
o
ÿ a
o
N
o
c
o
ÿ a
o
N
o
exp ÿ k
x
c
o
ÿ a
o
N
o
t
5
Equation 5 was used to ®t the entire data set pre-
sented in Fig. 1 by a least-square method. The
results from these eorts are presented in the ®gure
by the continuous lines. The corresponding ®tting
parameter values obtained were k
x
=1.7220.15 l/
(mg s) and a
o
=1.2920.0510
ÿ11
mg O
3
/CFU. As
depicted in Fig. 1, the second-order kinetic model
®tted the experimental data well for t<15 s.
Although deviations were observed for t>15 s,
these longer contact times were not relevant because
the E. coli inactivation reaction is much faster (k
i
/
k
x
180) and practically completed under 15 s for the
entire range of experimental conditions investigated.
TOC analyses of sonicated E. coli cell suspensions
at initial densities ranging from 6.8±1110
9
CFU/l
revealed that the average organic carbon content of
each
cell
was
approximately
TOC
o
/
N
o
=2.5620.1010
ÿ10
mg C/CFU. The ozone
demand per mass of organic carbon can be
expressed with the new parameter b
o
=a
o
N
o
/
TOC
o
=0.050420.0040 mg O
3
/mg C. The ozone
demand parameters could also be expressed on a
molecular base as a
o
=1.6210
8
O
3
molecules/CFU
and b
o
=0.0126 O
3
molecules/E. coli C atom.
Interestingly, the a
o
value for ozone was in reason-
ably good agreement with that of 710
7
molecules/
CFU for iodine demand by E. coli reported by
Scott and Lesher (1963). These authors proposed
that the oxidizing agent demand was exerted by car-
bon double bonds of unsaturated fatty acids present
in the cell wall and membrane. Assuming that each
ozone molecule reacts with a single carbon double
bond group, then the fraction of E. coli organic car-
bon participating in double bond groups would be
approximately 2.52% of the total carbon content.
It was assumed in equation 3 that the rate of
ozone disappearance in the presence of E. coli cells
can be represented by a homogeneous second-order
expression. The validity of this assumption was
checked by testing solutions containing both in-
itially viable, and sonicated E. coli cells. The results
from these experiments are presented in Fig. 2. A
somewhat faster ozone decay rate was observed for
the sonicated samples. As expected, predictions with
equation
3
using
k
x
=1.72 l/(mg s),
and
a
o
=1.2910
ÿ11
mg/CFU (continuous lines) were
closer to the data corresponding to the initially
viable cells. The slight over-predictions observed
might have resulted from a combination of analyti-
cal and experimental inaccuracies. In order to com-
pare these results, equation 3 was used to ®t the
data sets corresponding to the initially live and
sonicated cells (dashed lines). The resulting par-
ameters
were
k
x
=1.3320.14 l/(mg s)
and
a
o
=1.6620.0710
ÿ11
mg/CFU for initially live
cells
and
k
x
=2.3820.36 l/(mg s)
and
a
o
=1.7720.0810
ÿ11
mg/CFU for sonicated cells.
The fact that the a
o
values were comparable sup-
ported that the ultimate ozone demand was ap-
proximately the same for both types of cell
suspensions with the small dierence obtained likely
being the result of experimental variability. The
dierent k
x
values were indicative that the inacti-
vation process might have been mass transfer lim-
ited in agreement with disinfection mechanisms
based on reactivity with cell membrane components
(Christensen and Giese, 1954; Scott and Lesher,
1963). Accordingly, the faster reaction rate for soni-
cated cells indicated that ozone molecules reacted
faster with cell components. Consequently, the use
of equation 5 should be interpreted as a model
valid for quanti®cation but not necessarily an accu-
rate representation of the more complex mass trans-
fer steps and heterogeneous reactions taking place
during the inactivation process.
Experimental results obtained for the tests per-
formed to investigate the decomposition of ozone in
the presence of humic acid are presented in Fig. 3
for t<15 s. Equations 3±5 were used to ®t the
entire data set shown in the ®gure after substituting
Fig. 2. Apparent overall decomposition of ozone in the
presence of initially viable and sonicated E. coli for var-
ious initial ozone concentrations and micro-organism den-
sities (see set I in Table 1 for experimental conditions).
Nimrata K. Hunt and Benito J. MarinÄas
2636
x for y, and x
o
=a
o
N
o
for y
o
=b
o
TOC
o
. y is the fast
reacting portion of the TOC also expressed in mg/l
as O
3
, y
o
is the initial value for y, TOC
o
is the TOC
corresponding to the initial humic acid concen-
tration in solution, and b
o
is the initial fast ozone
demand by the humic acid tested also expressed in
mg O
3
/mg C. The ®tted curves were found to
match the experimental data reasonably well as
depicted in Fig. 3. The corresponding ®tting par-
ameters
were
k
y
=3.2020.16 l/(mg s)
and
b
o
=0.32420.005 mg O
3
/mg C. It should be
pointed out that similar to the observation for the
reaction with E. coli, the model deviated from the
experimental data for t>15 s possibly due to ozone
reactions with slower-reacting organic molecules
and radicals. However, as discussed earlier these
longer contact times were generally not relevant to
the inactivation process under the set of experimen-
tal conditions used.
The decomposition of dissolved ozone in the sim-
ultaneous presence of both E. coli and humic acid
can be represented by the expressions:
dc
dt
ÿk
x
cx ÿ k
y
cy
6
dx
dt
ÿk
x
cx
7
dy
dt
ÿk
y
cy
8
Equations 6±8 represent a system of independent
non-linear equations meeting the overall mass bal-
ance (c
o
ÿx
o
ÿy
o
)=(cÿxÿy) which can be used to
substitute any of the three expressions. The non-lin-
earity of the system of equations required solution
by an approximation approach. A ®nite dierence
approach was used with time interval Dt=0.003 s
or a total of 500 time elements within the range of
0±15 s. A trial-and-error approach revealed that
predictions were practically the same for lower Dt
values. Figure 4 presents a comparison of model
predictions to experimental data corresponding to
tests performed at N
o
=1.310
10
CFU/l, TOC
o
=0±
0.50 mg/l and c
o
=206±374 mg/l. The model par-
ameters were those independently determined by ®t-
ting the data in Figs. 1 and 3 (i.e. k
x
=1.72 l/(mg s),
k
y
=3.20 l/(mg s), a
o
=1.2910
ÿ11
mg O
3
/CFU and
b
o
=0.324 mg O
3
/mg C). As depicted in Fig. 4, the
model predicted the data well within the range of
experimental conditions investigated.
The inactivation of E. coli with ozone in the pre-
sence of natural organic matter such as humic acid
can be represented with the integrated form of
equation 1, or
ln
N
N
o
ÿk
i
t
0
c dt
9
where c changes with time according to equations
6±8. The results from inactivation experiments per-
formed with solutions containing humic acid at
concentrations TOC
o
=0.1±0.5 mg/l are shown in
Fig. 5 against the corresponding integrated ozone
exposure estimated from predicted curves similar to
those shown in Fig. 4. Also shown in Fig. 5 is the
line corresponding to an inactivation rate constant
of k
i
=138 l/(mg s) in the absence of humic acid esti-
Fig. 4. Apparent overall decomposition of ozone in the
simultaneous presence of initially viable E. coli and humic
acids for various initial ozone and total organic carbon
concentrations, and micro-organism densities (see set III
in Table 1 for experimental conditions).
Fig. 3. Apparent overall decomposition of ozone in the
presence of humic acids for various initial ozone and total
organic carbon concentrations (see set II in Table 1 for ex-
perimental conditions).
Inactivation of E. coli with ozone
2637
mated with equation 2 for the experimental tem-
perature of 208C (Hunt and MarinÄas, 1997). In gen-
eral, good agreement is observed between the
inactivation data in the presence of humic acid and
the line corresponding to the absence of natural or-
ganic matter.
A relationship can now be established between
the inactivation kinetics and the kinetics corre-
sponding to the reaction between molecular ozone
and the fast-reacting portion of E. coli cells:
N
N
o
x
x
o
k
i
k
x
10
Equation 10 was obtained by combining equation
9 with an expression of the same form resulting
from integrating equation 3. Values for the corre-
lation between N/N
o
and x/x
o
according to
equation 10 are presented in Table 2 together with
the corresponding predicted ozone exposure.
Approximately 2.8% loss of the fast-reacting con-
stituents of E. coli corresponded to 99% inacti-
vation of this micro-organism and by the time 25%
of the fast-reacting groups had reacted with molecu-
lar ozone, practically all micro-organisms present in
the solutions tested in this study (N
o
110
10
CFU/l)
were inactivated.
Representative micrographs obtained for the
TEM analyses are shown in Fig. 6. Corresponding
ozone initial concentration and exposure, exper-
imental and model predicted E. coli survival and
fast-ozone demand constituent residual fraction are
presented in Table 3. Cells representative of those
present in control samples not exposed to ozone are
shown in micrograph A. The various shapes shown
in the micrograph resulted from random orientation
of the rod-shaped E. coli cells embedded in the
epoxy resin. The lighter part of the cells was the
nuclear region containing some DNA ®brils and
electron-dense ribosomes, the latter appearing as
dark black dots (VanDemark and Batzing, 1987).
Cell capsules and outer membranes were easily dis-
tinguished but no distinct internal membrane was
visible. These observations were consistent with
those reported by Woldringh and Nanninga (1985)
for E. coli also ®xed ®rst with 2.5% glutaraldehyde
and then with 1% osmium tetroxide. These authors
indicated that this ®xation approach resulted in a
nucleoid with preserved disperse appearance with-
out the excessive precipitation of DNA ®brils result-
ing from the use of other ®xation solutions.
Micrograph B presents a sample treated for 30 s
with an initial ozone concentration of 9 mg/l. The
model described previously was applied to predict a
corresponding integrated exposure to ozone of
0.0826 mg s/l, an inactivation of approximately
99.999% and a fast-reacting group conversion of
13.2%. The predicted inactivation matched closely
the observed value. As shown in Fig. 6, the appear-
ance of cells in micrograph B, most of which have
lost viability, was not noticeably dierent from that
of the control.
Micrograph C in Fig. 6 corresponded to a sample
exposed to 0.175 mg s/l according to model predic-
tions. The model predicted survival ratio presented
in Table 3 should be considered more representative
than that observed which corresponded to approxi-
mately the detection limit of viable E. coli cells and
might have been aected by the occurrence of cell
clumps. Essentially all cells exposed to the con-
ditions corresponding to micrograph C were inacti-
vated. Compared to the two previous micrographs,
distinct changes were observed in the bacterial cells.
The nucleoid seemed to have contracted and a
coarse precipitation of DNA was apparent. An
eect similar to that observed in micrograph C has
been reported (Woldringh and Nanninga, 1985)
when the secondary structure of the DNA-binding
proteins is destroyed. Apparently, for the level ex-
posure corresponding to micrograph C, ozone was
Fig. 5. Inactivation of E. coli with ozone in the presence
of humic acids for various initial ozone and total organic
carbon concentrations and micro-organism densities (see
set IV in Table 1 for experimental conditions).
Table 2. Model prediction of micro-organism survival, fast ozone-
demand components residual, and corresponding integrated pro-
duct of ozone concentration and contact time
N/N
o
x/x
o
t
0
c dt
1
1
0
0.5
0.991
0.00502
10
ÿ1
0.972
0.0167
10
ÿ2
0.944
0.0334
10
ÿ3
0.917
0.0501
10
ÿ4
0.891
0.0667
10
ÿ5
0.866
0.0834
10
ÿ6
0.841
0.100
10
ÿ7
0.818
0.117
10
ÿ8
0.794
0.133
10
ÿ9
0.772
0.150
10
ÿ10
0.750
0.167
Nimrata K. Hunt and Benito J. MarinÄas
2638
Fig. 6. Transmission elctron micrographs of E. coli cells after exposure to ozone in a continuous-¯ow tubular reactor.
Operating conditions: pH 7.2; temperature=208C; N
o
=510
9
CFU/l; t=30 s; c
o
=0 mg/l (A), 9 mg/l (B), 18 mg/l (C) and
196 mg/l (D).
Table 3. Model prediction of micro-organism survival, fast ozone-demand components residual, and corresponding integrated product of
ozone concentration and contact time for the samples investigated by transmission electron microscopy (see Fig. 6)
Micrograph in Fig. 6
c
o
(mg/l)
t
0
c dt (mg s/l)$
N/N
o
x/x
o
observed
predicted
A
0
0
1
1
1
B
9
0.0826
110
ÿ5
1.1210
ÿ5
0.868
C
18
0.175
110
ÿ7
3.2510
ÿ11
0.740
D
197
4.21
<10
ÿ7
%
110
ÿ252
0.00071
E}
1000
28.1
<10
ÿ7
%
±%
10
ÿ21
$
Contact time t=30 s for all experiments.
%
No colonies formed.
}
Not shown in Fig. 6. A pellet could not be obtained by centrifugation.
Inactivation of E. coli with ozone
2639
able to penetrate the cells, and react with the pro-
teins, or the numerous enzymes involved in the con-
trol of DNA conformation in the nucleoid,
resulting in the precipitation of DNA. Another
major dierence between micrograph C and the two
previous pictures was the occurrence of more pro-
nounced convolutions of the cell envelope.
Micrograph D in Fig. 6 illustrates bacterial cells
after a predicted exposure to ozone of 4.21 mg s/l.
The overall density of the nuclear material was low
compared to the previous micrographs and the cell
envelopes appeared severely convoluted and hang-
ing loosely around the cell instead of ®tting snugly.
Some of the bacteria appeared to be disrupted, and
fragments of the lysed cells were observed. The
occurrence of cell lysis was further supported by the
fact that a pellet could not be obtained for a sus-
pension exposed to 28.1 mg s/l (sample E in Table
3).
The rate of E. coli inactivation with ozone is fast
compared to those for other micro-organisms of
public health interest such as protozoan parasites,
e.g. Cryptosporidium parvum. Drinking surface
water systems designed to inactivate C. parvum
oocysts must provide CT values several thousand
times greater than those required to protect against
E. coli (Rennecker et al., 1999). Consequently, the
E. coli inactivation kinetics developed in this study
might not have a direct impact on the design of
drinking water treatment plants. However, this in-
formation is of greater relevance in wastewater
treatment. For example, secondary wastewater eu-
ents from the Belmont and Southport Advanced
Wastewater Treatment Plants at Indianapolis, IN
are disinfected with ozone for the primary regulat-
ory compliance goal of reducing the density of
viable fecal coliforms (Blank et al., 1993).
Reductions in fecal coliforms achieved in the ozone
contactors of these plants are reported at 99±
99.99%, or N/N
o
=0.0001±0.01 (Blank et al., 1993).
These inactivation eciencies are low despite these
full-scale disinfection contactors being designed for
operation at applied ozone dosages of 6 mg/l and
t
10
contact time (time for 10% of tracer mass added
as a pulse-input immediately up-stream from the
contactor inlet to reach the euent) of 10 min
(Blank et al., 1993). Assuming that the average
fecal coliform resistance to ozone was the same as
that found in this investigation for E. coli and
neglecting the presence of bacterial clumps then
Fig. 5 can be used to estimate the CT values corre-
sponding to the observed N/N
o
at 0.03±0.07 mg s/l.
The average dissolved ozone concentrations corre-
sponding to these CT values can be estimated at
0.5±110
ÿ4
mg/l assuming that t
10
provided a good
estimate for the eective contact time achieved in
the contactors. As illustrated with this example, the
eciency of wastewater euent disinfection with
ozone is aected by ozone-demand reactions. If the
apparent decay rate of ozone in a certain waste-
water euent is known then a kinetic model similar
to that developed for this study could be integrated
with reactor mass transfer and hydrodynamic infor-
mation to predict the performance and perhaps
optimize the design of ozone disinfection units.
CONCLUSIONS
The apparent decomposition of dissolved ozone
in the presence of humic acid and E. coli cells was
modeled successfully with mixed second-order rate
expressions within a time scale relevant to E. coli
inactivation with ozone. Kinetic parameters
obtained for the separate reactions between ozone
and humic acid, and ozone and E. coli cells pre-
dicted well the overall decomposition of ozone in
the simultaneous presence of humic acid and E. coli
cells.
The rate for the ozone inactivation of E. coli with
ozone in the presence of humic acid was slower
than that in the absence of natural organic matter.
The dierence was due to the faster decomposition
of dissolved ozone and, thus, the lower exposure of
E. coli cells to the disinfectant. However, the sec-
ond-order inactivation rate constant was approxi-
mately the same in the presence and absence of
humic acid con®rming that dissolved ozone is the
species generally responsible for inactivation.
The overall chemical reaction between ozone and
organic matter in E. coli cells was slower that the
corresponding inactivation reaction. Only 25% of
the initial relatively fast ozone demand were satis-
®ed by the time that practically all micro-organisms
present in solution were inactivated. TEM micro-
graphs revealed that noticeable changes in the in-
terior of E. coli cells did not take place until most
of the cells present in the sample were non-viable.
Subsequent exposure to ozone resulted in structural
changes, membrane deterioration, and ultimately
lysis of the inactivated cells.
AcknowledgementsÐThe authors would like to thank Dr
Bruce Hunt, Dr Chung-Fan Chiou, Manalee Johnson and
Tom Cooper, School of Civil Engineering, Purdue
University, West Lafayette, IN for their technical assist-
ance and support at various stages of this study. TEM
analyses were performed by Dr John Turek and the lab-
oratory technicians at the School of Veterinary Medicine,
Purdue University, West Lafayette, IN. Financial support
by the City of Indianapolis, Department of Public Works,
Indianapolis, IN and the Schools of Engineering, Purdue
University, West Lafayette, IN are gratefully acknowl-
edged.
REFERENCES
Blank B. D., MarinÄas B. J., Corsaro K. and Rakness K.
L. (1993) Enhancement of wastewater disinfection e-
ciency in full-scale ozone bubble-diuser contactors.
Ozone Science and Engineering 15, 295±310.
Chick H. (1908) An investigation of the laws of disinfec-
tion. Journal of Hygiene 8, 92±158.
Nimrata K. Hunt and Benito J. MarinÄas
2640
Christensen E. and Giese A. C. (1954) Changes in absorp-
tion spectra of nucleic acids and their derivatives follow-
ing exposure to ozone and ultraviolet radiations.
Archives of Biochemistry and Biophysics 50, 206±209.
Hamelin C. and Chung Y. S. (1974) Optimal conditions
for mutagenesis by ozone in Escherichia coli K12.
Mutation Research 24, 271±279.
Hamelin C., Sarhan F. and Chung Y. S. (1977) Ozone
induced DNA degradation in dierent DNA polymerase
I mutants of Escherichia coli K12. Biochemical and
Biophysical Research Communication 77, 220±224.
Hamelin C., Sarhan F. and Chung Y. S. (1978) Induction
of deoxyribonucleic acid degradation in Escherichia coli
by ozone. Experentia 34, 1578±1579.
Hunt N. K. and MarinÄas B. J. (1997) Escherichia coli inac-
tivation with ozone. Water Research 31, 1355±1362.
Ishizaki K., Miura K. and Shinriki N. (1987) Eect of
ozone on plasmid DNA of Escherichia coli in situ.
Water Research 21, 823±827.
Perich J. R., McCammon L. R., Cronholm L. S.,
Fleischman M., Pavoni J. L. and Riesser V. (1975)
Inactivation kinetics of virus and bacteria in a model
ozone contacting reactor system. In Proceedings of the
Second International Symposium on Ozone Technology,
Montreal Canada, eds. R. G. Rice, P. Pichet and M.
Vincent, p. 486. International Ozone Institute, New
York, NY.
Pryor W. A., Dooley M. M. and Church D. F. (1983)
Mechanisms of the reactions of ozone with biological
molecules: the source of toxic eects of ozone. In
Advances in Modern Environmental Toxicology, eds. M.
G. Mustafa and M. A. Mehlman. Ann Arbor Science
Publishers, Ann Arbor, MI.
Rennecker J. L., MarinÄas B. J., Owens J. H. and Rice E.
W. (1999) Inactivation of Cryptosporidium parvum
oocysts with ozone. Water Research 33(11), 2481±2488.
Scott D. B. M. (1975) The eect of ozone on nucleic acids
and their derivatives. In Aquatic Application of Ozone,
eds. W. J. Blogoslawski and R. G. Rice, p. 1.
International Ozone Institute, New York, NY.
Scott D. B. M. and Lesher E. C. (1963) Eect of ozone on
survival and permeability of Escherichia coli. Journal of
Bacteriology 85, 567±576.
VanDemark P. J. and Batzing B. L. (1987) The Microbes:
an Introduction to their Nature and Importance.
Benjamin/Cummings Publishing Company, Menlo Park,
CA.
Watson H. E. (1908) A note on the variation of rate of
disinfection with the change in the concentration of dis-
infectant. Journal of Hygiene 8, 536±542.
Woldringh C. L. and Nanninga N. (1985) Nucleoid and
cytoplasm in the intact cell. In Molecular Cytology of
Escherichia coli, ed. N. Nanninga. Academic Press,
London, UK.
Zhou H. and Smith D. W. (1994) Kinetics of ozone disin-
fection in completely mixed system. J. Environ. Eng.
Div. ASCE 120, 841±858.
Inactivation of E. coli with ozone
2641