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Oxygen and photoinactivation of Escherichia coli in UVA andsunlight
R. Khaengraeng and R.H. ReedDivision of Biomedical Sciences, Northumbria University, Newcastle upon Tyne, UK
2003/0711: received 15 August 2003, revised and accepted 11 January 2005
ABSTRACT
R. KHAENGRAENG AND R.H. REED. 2005.
Aims: To establish the influence of oxygen on Escherichia coli before, during and after exposure to UVA or
simulated sunlight.
Methods and Results: Bacterial suspensions were exposed either to UVA or simulated sunlight. Conventional
aerobic plate counts of illuminated cell suspensions were consistently lower than those obtained under conditions
where reactive oxygen species (ROS) were neutralized, either (i) by the addition of the peroxide scavenger sodium
pyruvate (0Æ05% w/v) to the medium with subsequent incubation in an anaerobic jar or (ii) by culturing on a
prereduced medium within an anaerobic cabinet, indicating that a substantial proportion of such cells are sublethally
injured. While the presence of oxygen during the growth period resulted in a greater resistance of aerobically grown
cells to simulated sunlight compared with their anaerobic counterparts, the extent of inactivation during
illumination was directly related to the dissolved oxygen content of the water.
Conclusions: The results show that, at each stage, oxygen has a marked influence on the observed colony count.
Significance and Impact of the Study: Overall, the results indicate that future studies of bacteria exposed to
UVA or sunlight should consider the effects of oxygen at every stage in the procedure, and especially during
enumeration, where the inhibitory effects of ROS must be neutralized in order to obtain a valid count. An
investigation of the effects of ROS neutralization on the counts of faecal bacteria under field conditions in natural
waters is now required to establish the significance of these finding to solar water treatment.
Keywords: anaerobiosis, E. coli, photo-oxidation, pyruvate, solar disinfection, sunlight.
INTRODUCTION
While the antimicrobial properties of light have been known
for well over a century (Downes and Blunt 1877), more
recent research has focussed on the effects of sunlight on the
survival of bacteria in natural waters. This is particularly
important with regard to the persistence of faecal indicator
bacteria, e.g. Escherichia coli (Alkan et al. 1995) and
Enterococcus faecalis (Sinton et al. 2002), and water-borne
enteric pathogens such as Shigella flexneri (Runyen-Janeckyet al. 1999) and Vibrio cholerae (Oufdou et al. 2000). Many
research studies have suggested that sunlight is one of the
most important factors responsible for the inactivation of
faecal bacteria in fresh water (e.g. Barcina et al. 1990) and in
sea water (e.g. Davies and Evison 1991; Sinton et al. 1999).The disinfection of drinking water using sunlight was first
proposed by Acra et al. (1980) for the treatment of oral
rehydration solutions and subsequently demonstrated to be
effective against a range of bacteria, using laboratory isolates
(e.g. Wegelin et al. 1994; Joyce et al. 1996; Walker et al.2004) and naturally contaminated waters (e.g. Lawand et al.1997; Sommer et al. 1997; Caslake et al. 2004). Most of the
inactivation is as a result of solar UVA radiation, accounting
for c. 70% of the negative effects of sunlight (Acra et al.1984). Solar water treatment has considerable potential as a
low-cost means of treating contaminated drinking water in
developing countries with consistently sunny climates
Correspondence to: R.H. Reed, Division of Biomedical Sciences, School of Applied
Sciences, Northumbria University, Ellison Place, Newcastle upon Tyne, Tyne and
Wear NE1 8ST, UK (e-mail: [email protected]).
ª 2005 The Society for Applied Microbiology
Journal of Applied Microbiology 2005, 99, 39–50 doi:10.1111/j.1365-2672.2005.02606.x
(McGuigan et al. 1999), and was highlighted as an effective
small-scale method for the improvement of drinking water
quality on World Water Day 2001 (Anon 2001).
Although the early studies of solar disinfection took no
account of the level of aeration of the water to be treated,
subsequent research has shown that a high level of dissolved
oxygen is required for the rapid inactivation of Escherichiacoli and Enterococcus faecalis suspended in distilled water and
then exposed to sunlight (Reed 1996, 1997). Similar results
have been reported for the effects of oxygen on solar
disinfection of faecal coliforms and faecal streptococci under
field conditions (Reed et al. 2000; Meyer and Reed 2001).
Taken together, these studies have shown that the rate of
inactivation of faecal bacteria exposed to sunlight is four to
eight times faster in oxygenated water compared with hypo-
oxygenated water, demonstrating that photo-oxidation is the
principal reason for the rapid decrease in bacterial counts.
The underlying process responsible for this inactivation is
the light-dependent production of reactive forms of oxygen,
including oxygen-free radicals such as superoxide and
hydroxyl radicals (Gourmelon et al. 1994), along with toxic
derivatives such as hydrogen peroxide (Alam and Ohgaki
2002). These reactive oxygen species (ROS) are generated
mainly as a result of the absorption of light by endogenous
photosensitizers, e.g. intracellular porphyrins and flavins
(Curtis et al. 1992), although exogenous photosensitizers
may also be significant in the generation of ROS in natural
waters (Voelker et al. 1997; Davies-Colley et al. 1999).To date, most studies of solar disinfection have been
carried out using isolates of E. coli enumerated by culture on
conventional selective or nonselective agar-based media,
incubated under standard aerobic conditions (e.g. Clesceri
et al. 1998; Anon 2002). However, recent studies have
shown that, while such conditions are effective in enabling
the growth of healthy cells, they do not always allow
physiologically damaged cells to grow. Thus, suspensions of
heat-damaged E. coli O157 cells have been found to give
higher counts in growth media cultured under anaerobic
conditions, compared with aerobic incubation (Murano and
Pierson 1992; Bromberg et al. 1998). Such effects are also
modulated by the prior growth of the bacteria under aerobic
or anaerobic conditions (George et al. 1998). Similar effects
have been observed for other bacteria, including Salmonella(Xavier and Ingham 1993) and Listeria (Knabel and Thielen
1995; Gnanou Besse 2002). Other studies have demonstrated
that higher counts can be obtained for heat-injured E. coli byincorporating substances that interact with reactive forms of
oxygen into agar-based growth media; these substances
include scavengers of peroxides such as pyruvate (e.g.
Czechowitz et al. 1996) and dissimilatory enzymes such as
catalase (Mackey and Seymour 1987), supporting the
proposal that ROS may inhibit the growth of sublethally
injured cells of E. coli under aerobic conditions.
The present study was carried out to investigate the effects
of oxygen status on the inactivation of E. coli exposed to either(i) UVA or (ii) simulated full-spectrum sunlight, by compar-
ing conventional plate counts on a nonselective, agar-based
medium for cells grown, illuminated and incubated either in
the presence or the absence of oxygen. The results show that,
at each stage, i.e. during (i) initial culture, (ii) illumination and
(iii) subsequent enumeration, oxygen had a marked influence
on the final colony count, showing a positive effect during
initial culture, and a negative effect during exposure to light
and in the postillumination enumeration stage. This was
particularly evident in relation to the negative effects of
oxygen (via ROS) on cells sublethally injured by photo-
oxidation, with maximum colony counts obtained only under
conditions where toxic metabolites of oxygen were subse-
quently prevented from inhibiting growth, e.g. by using a
combination of a growth medium with added pyruvate
together with incubation in an anaerobic jar, or by cultivation
on a prereduced medium within an anaerobic cabinet.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Escherichia coli NCTC8912 was obtained from the National
Collection of Type Cultures (Colindale, UK). Stock cultures
of this strain were maintained by repeated subculture on
nutrient agar (Oxoid, Basingstoke, UK) at 37�C, and used formost of the experiments described in the present study. Four
other isolates were used in comparative experiments: E. coliNU9904 and NU9906 (NU, Northumbria University Strain
Collection), both obtained from water from the River Tyne at
Howden; NU9912, obtained from a urine sample; and
NCTC12900, a nontoxigenic strain of E. coli O157. Standardexperimental cultures were prepared by loop inoculation of
20 ml of sterile nutrient broth (Merck VWR, Darmstadt,
Germany) in a Universal bottle (25 ml volume), followed by
18 h incubation at 37�C without shaking. The broth cultures
reached stationary phase at 18 h (determined spectrophoto-
metrically at 550 nm, A1cm ¼ 1Æ2) under anaerobic condi-
tions, with no oxygen detectable using an oxygen probe
(model 9010; Jenway, Dunmow, UK). Anaerobic conditions
at 18 h were also confirmed by the decolourization of a dilute
solution of the redox dye methylene blue to the broth culture.
For experiments where only fully oxygenated water was
required, suspensions of bacteria were shaken for 2 min
before illumination, to ensure that air equilibration and
oxygen saturation was achieved. To investigate the effects of
oxygen status during illumination, sterile water was sparged
either with sterile air (oxygenated conditions, with O2
measured at 7Æ5 mg l)1), sterile oxygen-free N2 gas (hypo-
oxygenated conditions, O2 at 0Æ9 mg l)1) or sterile O2 gas
(hyperoxygenated conditions, O2 at 27Æ2 mg l)1) for 1 h
40 R. KHAENGRAENG AND R.H. REED
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
prior to the addition of E. coli (all gases were obtained from
BOC Special Gases, London, UK). The same treatments
were then continued throughout the illumination period, to
maintain the appropriate level of oxygenation during the
experiment, with measured pH values of 6Æ2 (air-sparged),
6Æ6 (N2-sparged) and 6Æ7 (O2-sparged) at the end of the
experiment, compared with an initial pH value of 6Æ1.For a direct comparison of aerobically grown and
anaerobically grown cultures, nutrient broths were sparged
either with sterile air or with sterile oxygen-free N2 gas
throughout the growth period. Cultures were first grown
overnight at 37�C to stationary phase (A550 ¼ 1Æ2) with
sparging using either air or N2, then subcultured to fresh
nutrient broth and incubated at 37�C to early log phase
(A550 ¼ 0Æ2) under the same conditions, to give suspensions
of metabolically active cells.
Preparation and illumination of cell suspensions
Broth cultures were centrifuged at 12 000 g for 1 min (MSE
Microcentaur; Fisher Scientific, Loughborough, UK), re-
suspended in sterile distilled water and recentrifuged at
12 000 g for 1 min. The resuspension and recentrifugation
was repeated, to remove all traces of the original growth
medium from the cells and thereby remove any potential
exogenous photosensitizers and/or medium components
that might restrict light penetration. The cells were then
resuspended in 5 ml of sterile distilled water at a density of
107–108 CFU ml)1, placed in a 10-ml borosilicate glass tube
and exposed to light from either (i) a UVA source (40W-R
sunlamp tubes; Philips, Croydon, UK), at a UVA irradiance
of 20 W m)2, measured with a SKU421/I UVA sensor and
SKT660 meter (Skye Instruments, Llandrindod Wells,
UK) or (ii) a 1000-W solar simulator (Model 91190 large
area light source; Oriel, Stanford, CA, USA) at a total
irradiance of 410 W m)2, measured using an SKS1110
pyranometer and SKT660 meter (Skye Instruments, Lland-
rindod Wells, UK). While the UVA source provides a broad
band of UVA light, with maximum emission in the range
360–380 nm (Khaengraeng 2004), the solar simulator pro-
vides a reasonable fit to the whole solar spectrum from UV
to infrared wavelengths (Kehoe 2001). The ambient tem-
perature during illumination was 24–26�C and the tubes
were cooled by fan throughout their exposure to the light
source, to prevent excessive temperature rises during
experimentation (the temperature during illumination never
exceeded 32�C), to minimize thermal effects (McGuigan
et al. 1998).
Enumeration
Timed samples were taken from the illuminated cell
suspensions and immediately processed in a dimly lit room
(irradiance, <5 W m)2), to avoid further photoinactivation.
The samples were subject to serial decimal dilution in
quarter-strength Ringer’s solution, then surface spread
(0Æ02–0Æ1 ml, depending on sample, dilution and time) onto
Petri plates containing freshly prepared nutrient agar
(Oxoid), as before (Reed 1997). Chemical supplements
(ROS-scavenging agents) required in particular experi-
ments, i.e. ascorbate, cysteine, dithiothreitol, histidine,
mannitol and pyruvate, were added to the nutrient agar
before autoclaving at 121�C for 15 min. In contrast, the
enzymes catalase and superoxide dismutase, were added to
autoclaved molten agar cooled to 50�C, prior to pouring
because of their heat lability at higher temperatures. All
supplements were obtained from Sigma Aldrich (Gilling-
ham, UK).
Inoculated media were incubated either aerobically or
anaerobically in a 2Æ5-l anaerobic jar (Anaerocult�, with
Anaerocult� A Sachet; Merck VWR) at 37�C under
conditions of total darkness, either in a conventional aerobic
incubator (model MIR-153; Sanyo, Osaka, Japan), or at
37�C in an anaerobic cabinet (Bugbox controlled environ-
ment workstation; Fred Baker Scientific, Runcorn, UK)
under an atmosphere of 10% H2 : 10% CO2 : 80% N2
(standard anaerobic gas mixture; BOC Special Gases,
Guildford, UK). Counts were performed using a colony
counter (Stuart Scientific, Redhill, UK) after 48 h, and then
checked at daily intervals thereafter, up to 7 d; colony
counts of appropriate dilutions were converted to CFU ml)1
by correcting for dilution and volume (Reed 1997). Samples
were counted in triplicate; counts are expressed as geometric
means of log-transformed data, with 95% confidence limits.
All graphs show the lowest detectable count (10 CFU ml)1,
log transformed to 1) as the minimum point on the y-axis.Where appropriate, paired t-tests have been used for
comparison of geometric means (McFeters et al. 1986):
statistically significant differences are noted where P < 0Æ05.The time for a 90% reduction in count (T90) was calculated
from plate counts of illuminated samples, as:
T90 ¼ t2 � t1logNt1 � logNt2
;
where Nt1 and Nt2 are initial and final counts at times t1 andt2 respectively. T90 values are quoted to the nearest minute.
RESULTS
Effect of oxygen scavengers and oxygen status onthe growth of UVA-treated and solar-illuminatedE. coli
Counts are shown in Fig. 1a for stationary phase E. coliNCTC8912 following harvesting and suspension in sterile
distilled water but prior to UVA exposure, plated onto
PHOTOINACTIVATION OF E. COLI 41
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
nutrient agar with different amounts of added sodium
pyruvate (a scavenger of peroxides; McDonald et al. 1983)and then cultured either aerobically or in an Anaerocult�anaerobic jar for 48 h. A similar count was obtained in all
treatments, irrespective of whether the plates were incuba-
ted aerobically or anaerobically, or whether the medium was
supplemented with pyruvate or not. This pattern was also
observed in all subsequent experiments (data not shown),
confirming that the initial inoculum of active, healthy cells
was not influenced significantly either by oxygen status, or
by peroxide scavengers in the medium.
Figure 1b shows counts for the same suspension of E. coliNCTC8912 following 2 h exposure to UVA in air-equili-
brated (oxygenated) water. Overall, the counts were lower
than for the initial inoculum, confirming the inhibitory
effects of UVA exposure. However, the count obtained was
strongly dependent upon the incubation conditions: under
aerobic conditions, the addition of pyruvate at 0Æ01% w/v
and above caused a statistically significant increase (t-test:
P < 0Æ05) in the observed count, with no apparent differ-
ences between counts made with added pyruvate at 0Æ01–0Æ5% w/v. Similarly, under anaerobic conditions (anaerobic
jar), all of the pyruvate-supplemented media showed
a statistically significant higher count, compared with
the unsupplemented (pyruvate-free) anaerobic medium,
although some evidence of a reduction in efficacy was
observed at the highest concentration of pyruvate (0Æ5%w/v) under anaerobic conditions. It should also be noted
that the UVA-treated cells grown on pyruvate-supplemen-
ted medium showed a statistically significantly higher count
under anaerobic conditions than under aerobic conditions at
all levels of pyruvate between 0Æ01 and 0Æ1% w/v. Further
incubation of the plates beyond 48 h, gave no substantial
increase in colony counts on any medium, in this and in all
subsequent experiments, with only the occasional additional
colony observed on one or two plates up to 7 d. This
demonstrates that 48 h incubation was sufficient to provide a
valid count for UVA-treated cells (note that 48 h incubation
was required in order to allow colonies to grow under
anaerobic conditions to a size large enough to be counted).
The results of an experiment carried out to establish the
effect of added catalase on the same batch of cells of E. coliNCTC8912 exposed to UVA for 2 h in fully oxygenated
water are shown in Fig. 2, demonstrating a statistically
significant increase in the count at 10 U plate)1 added
catalase and above under aerobic and anaerobic conditions,
compared with their respective unsupplemented controls.
As with pyruvate, all levels of catalase supplementation gave
a significantly higher count under anaerobic conditions,
compared with the corresponding aerobic treatment. A
further experiment showed no difference between the
1
3
5
7
9
0 0·01 0·05 0·1 0·5
Pyruvate (% w/v)
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
1
3
5
7
0 0·01 0·05 0·1 0·5
Pyruvate (% w/v)
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
Fig. 1 Effects of sodium pyruvate on enumeration of UVA-exposed
Escherichia coli. Geometric mean counts (log10 CFU ml)1) for a
suspension of stationary phase E. coli NCTC8912 in water (a) prior to
UVA exposure and (b) following 2 h exposure to UVA, cultured on
nutrient agar under aerobic conditions (open bars) or in an anaerobic
jar (grey bars) at different levels of added sodium pyruvate, up to 0Æ5%w/v. The initial inoculum, prior to UVA exposure, represents an
overall average count of 7Æ5 · 107 CFU ml)1. Error bars represent
95% confidence limits (n ¼ 3)
1
3
5
7
0 10 50 100 500
Catalase (U plate–1)
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
Fig. 2 Effects of catalase on enumeration of UVA-exposed Escherichia
coli. Geometric mean counts (log10 CFU ml)1) for a suspension of
stationary phase E. coli NCTC8912 in water following 2 h exposure to
UVA, cultured on nutrient agar under aerobic conditions (open bars)
or in an anaerobic jar (grey bars) at different levels of added catalase up
to 500 U plate)1. The initial inoculum in this experiment (0 h UVA
exposure) was 7Æ5 · 107 CFU ml)1. Error bars represent 95% con-
fidence limits (n ¼ 3)
42 R. KHAENGRAENG AND R.H. REED
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
combined addition of pyruvate plus catalase and supple-
mentation with either component alone (data not shown).
This is consistent with the similar effect of pyruvate and
catalase, as the former detoxifies hydrogen peroxide by
reacting to form acetate, water and CO2 (Elstner and Heupel
1976) while the latter enzymically converts hydrogen
peroxide to water and O2 (Mongkolsuk and Helmann 2002).
Calculation of T90 values for the pyruvate supplementa-
tion experiment (Fig. 1) showed that aerobic incubation in
pyruvate-free medium gave the lowest T90, at 32 min, rising
to 81 min under anaerobic conditions with pyruvate
supplementation at 0Æ05 and 0Æ1% w/v. Similarly T90 values
for the catalase supplementation experiment (Fig. 2) also
gave a value of 84 min under anaerobic conditions with
500 U plate)1 added catalase, which is broadly equivalent to
the equivalent value for pyruvate-supplemented medium.
Experiments were performed by adding scavengers of
other ROS to nutrient agar, including mannitol (a scavenger
of hydroxyl-free radicals; Shen et al. 1997) at up to 1Æ0%w/v, histidine (a quencher of singlet oxygen; Singh and
Kshatriya 2002) at up to 1Æ0% w/v, superoxide dismutase
(which enzymically detoxifies superoxide-free radicals) at up
to 200 U plate)1, the antioxidant ascorbate (Potters et al.2002) at up to 0Æ5% w/v and the reducing agents cysteine
and dithiothreitol (Suh and Knabel 2000; Nebra et al. 2002)at up to 0Æ1% w/v. None of these supplements showed a
statistically significant change in the count of UVA-treated
E. coli NCTC8912 under either aerobic or anaerobic
conditions (data not shown). Consequently, it was decided
to focus only on pyruvate supplementation at 0Æ05% w/v in
all further experiments, as this amount had a positive effect
under aerobic and anaerobic conditions (Fig. 1) and because
this concentration is consistent with earlier studies (e.g.
Sartory and Howard 1992). Sodium pyruvate was selected in
preference to catalase as the latter is heat-sensitive and
cannot be added prior to autoclaving.
Figure 3 shows data for stationary phase E. coliNCTC8912 exposed to UVA for 4 h and then cultured on
nutrient agar in the presence or absence of 0Æ05% w/v
pyruvate either aerobically, or under one of three different
sets of anaerobic conditions, to see the effects on the counts
obtained. For the first two sets of anaerobic plates, following
dilution and spread plating on the laboratory bench in aerobic
conditions, the plates were then transferred either to an
anaerobic jar, which takes c. 1 h to create fully anaerobic
conditions (Anon 2000) or to an anaerobic cabinet, giving a
fully anaerobic atmosphere from the outset. The third set of
anaerobic plates was prepared entirely within the anaerobic
cabinet, by spread plating onto prereduced media (kept for
48 h in the anaerobic cabinet prior to use), with incubation in
the same cabinet, to maintain anaerobic conditions through-
out the preparation phase and culture period. As in the
previous experiment, the results show that there was a
substantial increase in the count observed for the pyruvate-
supplemented plates under aerobic conditions. A smaller
effect of pyruvate supplementation was seen for plates
prepared in air and then transferred to the anaerobic jar
and the anaerobic cabinet while no significant difference was
observed between pyruvate-supplemented and unsupple-
mented plates in the anaerobic cabinet when prereduced
plates were used (Fig. 3). These results indicate that a
combination of pyruvate and an anaerobic jar can give a count
directly equivalent to that obtained with an anaerobic cabinet
and the prereduced growth media. An anaerobic jar, with
initial dilution and plating in air, was used in all subsequent
experiments, as an anaerobic cabinet was not readily available.
Table 1 shows data for four other strains of E. coli grownto stationary phase and then exposed to UVA for 2 h and
counted aerobically and anaerobically in the presence and
absence of 0Æ05% w/v pyruvate. In general, the results were
similar to those obtained using E. coli NCTC8912 (Fig. 1),
with a high count obtained under anaerobic conditions with
0Æ05% added pyruvate. The greatest proportional increase
was seen for UVA-treated E. coli NU9912, which gave a
count of 8Æ3 · 103 CFU ml)1 under aerobic conditions and
3Æ1 · 105 CFU ml)1 anaerobically with added pyruvate.
The latter value is over 30 times higher than the former,
indicating that anaerobic incubation in a medium with added
pyruvate can give a substantially higher count for some
strains. UVA-treated E. coli NU9904 showed a lower
reduction in count when assayed under aerobic conditions
(1Æ25 · 106 CFU ml)1) and a far smaller effect either of
1
2
3
4
5
AIR AJ AC AC-pre
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
Fig. 3 Effects of various combinations of ROS-neutralized conditions
on enumeration of UVA-exposed Escherichia coli. Geometric mean
counts (log10 CFU ml)1) for a suspension of stationary phase E. coli
NCTC 8912 in water following 4 h exposure to UVA, then cultured
either on unsupplemented nutrient agar (open bars) or on nutrient agar
with 0Æ05% w/v sodium pyruvate (grey bars) under aerobic conditions
(AIR), transferred from aerobic conditions to either an anaerobic jar
(AJ) or an anaerobic cabinet (AC), or prepared and cultured in an
anaerobic cabinet on prereduced nutrient agar (AC-pre). The initial
inoculum in this experiment (0 h UVA exposure) was c.
4Æ4 · 107 CFU ml)1. Error bars represent 95% confidence limits
(n ¼ 3)
PHOTOINACTIVATION OF E. COLI 43
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
anaerobic incubation and pyruvate addition, with an increase
in count to 1Æ75 · 106 CFU ml)1, representing an increase
of less than half of the aerobic value (note also that the values
for pyruvate-supplemented medium are almost the same
under aerobic and anaerobic conditions). NU9906 showed a
substantially higher count following anaerobic incubation
than for plates incubated aerobically, but with little evidence
of any further effect of pyruvate supplementation, suggest-
ing that individual strains may respond differently to this
combination of conditions. The consequent effects on
apparent T90 values are also shown in Table 1, with UVA-
treated E. coli NU9912 showing an increase in T90 of over
100% when the data for aerobic and anaerobic plus pyruvate
counts are compared, while E. coli NU9904 showed an
increase of c. 10% under the same conditions.
Effect of pyruvate and anaerobiosis on theapparent inactivation of E. coli exposed tosimulated sunlight
Figure 4 shows a time course for stationary phase E. coliNCTC8912 illuminated in the solar simulator in fully
oxygenated water for up to 6 h. The trend line for each set
of incubation conditions is close to a linear relationship, with
no evidence of an initial plateau or �shoulder�. The differencein count between the various incubation conditions shows an
increase with exposure time, being minimal at the start of
the experiment and increasing thereafter, up to 6 h, with the
aerobic count being lowest and a combination of anaerobic
incubation plus pyruvate supplementation giving the highest
count. Thus, by the end of the illumination period, the
disparity in counts between the unsupplemented plates
incubated aerobically and the pyruvate-supplemented plates
incubated anaerobically was almost exactly 100-fold. In
contrast, pyruvate-supplementation under aerobic condi-
tions gave a count of around half of that on the same
medium under anaerobic conditions, showing that the
addition of pyruvate alone is not able to counteract all of
the inhibitory effects of ROS. Similar effects have also been
observed following exposure to natural sunlight in PET
bottles containing 1 l of bacterial suspension, with the
highest counts obtained on pyruvate-supplemented medium
incubated in an anaerobic jar (data not shown).
Calculating the apparent T90 values for each set of
incubation conditions using the data for 0 and 6 h shown in
Fig. 4 gives a value of 95 min for aerobic incubation of
unsupplemented medium, 160 min for aerobic incubation of
pyruvate-supplemented medium, 175 min for anaerobic
incubation of unsupplemented medium and 200 min for
anaerobic incubation of pyruvate-supplemented medium,
the latter being more than double the value for standard
aerobic incubation, in broad agreement with the results for
UVA-treated cells.
Effects of oxygen status during growth andillumination with simulated sunlight on theapparent inactivation of E. coli
A broth culture of E. coli NCTC8912 was grown to early log
phase under oxygenated (air-sparged) or hypo-oxygenated
Table 1 Comparative inactivation of four
different isolates of Escherichia coli. Geometric
mean counts (log CFU ml)1) of cells sus-
pended in water and exposed to UVA illu-
mination for 2 hStrain
Initial count
aerobic
Count following UVA exposure
Aerobic
Aerobic +
0Æ05% w/v
pyruvate Anaerobic
Anaerobic +
0Æ05% w/v
pyruvate
NU9904 7Æ57 6Æ10 (82) 6Æ23 (90) 6Æ14 (84) 6Æ24 (90)
NU9906 7Æ84 6Æ42 (85) 6Æ53 (92) 7Æ06 (154) 7Æ03 (148)
NU9912 7Æ54 3Æ92 (33) 5Æ04 (48) 4Æ93 (46) 5Æ49 (76)
NCTC12900 7Æ65 4Æ51 (38) 5Æ12 (47) 4Æ61 (39) 5Æ42 (54)
T90 values (min) are given within parenthesis.
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Time (h)
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
Fig. 4 Time course for apparent inactivation of Escherichia coli
exposed to simulated sunlight and enumerated under various combi-
nations of ROS-neutralized conditions. Geometric mean counts (log10CFU ml)1) for a suspension of stationary phase E. coli NCTC8912 in
water exposed to simulated sunlight for up to 6 h and cultured either
aerobically (open symbols) or anaerobically (closed symbols) on
unsupplemented nutrient agar (circles) or on nutrient agar containing
0Æ05% w/v sodium pyruvate (squares). Error bars represent 95%
confidence limits (n ¼ 3)
44 R. KHAENGRAENG AND R.H. REED
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
(N2-sparged) conditions and then illuminated in the solar
simulator under either hypo-oxygenated (N2-sparged), oxy-
genated (air-sparged) or hyperoxygenated (O2-sparged)
conditions. Samples of treated cells were then counted
either under aerobic conditions in unsupplemented nutrient
agar or under anaerobic conditions (anaerobic jar) in
pyruvate-supplemented medium, as the latter consistently
gave the highest counts in all the earlier experiments.
Figure 5 shows the data for log phase cells grown with N2
sparging. Comparing the results for plates of unsupple-
mented medium incubated aerobically, it is clear that
illumination with simulated sunlight in hyperoxygenated
(O2-sparged) water resulted in a rapid decrease in counts,
with no evidence of an initial delay or �plateau� when
compared with oxygenated (air-sparged) conditions, with
the hypo-oxygenated treatment showing the clearest evi-
dence of a plateau and the slowest overall decrease in counts.
Such results are consistent with the earlier observation that
the extent of photoinactivation is directly related to the level
of dissolved oxygen in the water during illumination (Curtis
et al. 1992; Reed 1997), rather than being linked to the
relatively small changes in pH observed as a result of
sparging. Similarly, the results for anaerobic enumeration
using pyruvate-supplemented medium show the same
overall trend, although in each case, the overall decrease at
any time point was less than that observed under aerobic
conditions, with greater evidence of an initial plateau. This
plateau effect is most clearly seen for the cell suspension
illuminated in hypo-oxygenated water, where the decrease
was most marked following 3 h illumination, when counted
anaerobically with pyruvate, in contrast to that observed
aerobically in unsupplemented medium. Comparing the two
sets of incubation conditions for each of the three
treatments, the smallest differences were seen for the
illuminated O2-sparged cells, while the largest disparities
were seen for N2-sparged cells where the aerobic count in
unsupplemented medium dropped below the detection limit
of 10 CFU ml)1 after 4 h but remained countable up to 6 h
under anaerobic conditions in pyruvate-supplemented
medium.
Figure 6 shows the corresponding data set for early log
phase cells of E. coli NCTC8912 grown under air-sparged
(oxygenated) conditions. In general, a similar overall trend
was seen to that observed for the cells cultured under N2,
with (i) cells illuminated under hyperoxygenated conditions
showing the fastest decrease, followed by oxygenated and
then hypo-oxygenated conditions and (ii) counts made in
pyruvate-supplemented medium under anaerobic conditions
consistently showing higher values than the corresponding
aerobic counts in unsupplemented medium. When Fig. 6 is
compared with Fig. 5, it is clear that the aerobically grown
cells were more resistant to illumination in simulated
sunlight than cells cultured under hypo-oxygenated condi-
tions, as each of the curves shown in Fig. 6 decreases at a
slower rate than that of the equivalent data set in Fig. 5.
Additionally, the differences between aerobic, unsupple-
mented counts and anaerobic, pyruvate-supplemented
counts were smaller for cells grown with air sparging,
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Time (h)
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
Fig. 5 Time course for apparent inactivation of Escherichia coli grown
under hypo-oxygenated conditions and illuminated under different
levels of oxygen. Geometric mean counts (log10 CFU ml)1) for a
suspension of E. coli NCTC8912 grown to early log phase under hypo-
oxygenated conditions (N2-sparged), then suspended in water and
exposed to simulated sunlight for up to 6 h either under hypo-
oxygenated (N2-sparged) conditions (circles), oxygenated (air-sparged)
conditions (squares) or hyperoxygenated (O2-sparged) conditions
(triangles), and counted either on unsupplemented medium under
aerobic conditions (open symbols) or on medium supplemented with
0Æ05% w/v sodium pyruvate under anaerobic conditions (filled
symbols). Error bars represent 95% confidence limits (n ¼ 3)
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Time (h)
Pla
te c
ount
(lo
g 10
CF
U m
l–1)
Fig. 6 Time course for apparent inactivation of Escherichia coli grown
under oxygenated conditions and illuminated under different levels of
oxygen. Geometric mean counts (log10 CFU ml)1) for a suspension of
E. coli NCTC8912 grown to early log phase under oxygenated
conditions (air-sparged), then suspended in water and exposed to
simulated sunlight for up to 6 h either under hypo-oxygenated (N2-
sparged) conditions (circles), oxygenated (air-sparged) conditions
(squares) or hyperoxygenated (O2-sparged) conditions (triangles), and
counted either on unsupplemented medium under aerobic conditions
(open symbols) or on medium supplemented with 0Æ05% w/v sodium
pyruvate under anaerobic conditions (filled symbols). Error bars
represent 95% confidence limits (n ¼ 3)
PHOTOINACTIVATION OF E. COLI 45
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
compared with N2 sparging, especially in the early stages of
illumination, supporting the idea that cells grown under
aerobic conditions are less sensitive to the subsequent
damaging effects of ROS, presumably as a result of their
antioxidant defence systems. These differences are also
evident when the T90 values for each treatment are
calculated (Table 2): cells under hypo-oxygenated condi-
tions had lower T90 values under all enumeration conditions,
compared with the corresponding aerobically grown cells,
with the greatest differences being observed on illumination
under hypo-oxygenated conditions and with smaller differ-
ences for the hyperoxygenated treatment. Overall, the T90
values for illuminated cells counted anaerobically in pyru-
vate-supplemented medium are 30–72% greater than the
corresponding unsupplemented aerobic values, with the
greatest proportional increases being seen for cells illumin-
ated under hypo-oxygenated conditions.
DISCUSSION
The enhanced enumeration of injured bacteria by peroxide-
degrading compounds such as catalase and pyruvate is well
established, and pyruvate is often incorporated into con-
ventional culture media for aerobic incubation of food and
water samples, e.g. Baird-Parker agar (Schoeller and
Ingham 2003), mLSA medium (Sartory 1995) and R2A
medium (Reasoner and Geldrich 1985). Bogosian et al.(2000) have shown that cold-stored Vibrio vulnificus can give
counts up to 1000-fold higher on aerobic media containing
catalase or pyruvate compared with unsupplemented con-
trols, and they attributed this increase to the degradation of
peroxides produced during autoclaving of the growth
medium. In contrast, Mizunoe et al. (1999) attributed
similar effects of pyruvate and catalase in temperature-
stressed E. coli O157 to the metabolic production of
hydrogen peroxide. The results in Figs 1–6 show that
supplementation of nutrient agar with 0Æ05% pyruvate can
give substantially higher aerobic counts for E. coli exposedto UVA or simulated sunlight, in agreement with the
observations of Kehoe (2001) for solar-illuminated bacteria,
including E. coli, S. flexneri and V. cholerae. However, the
current study also demonstrates that such counts are further
enhanced when pyruvate-supplemented plates are trans-
ferred to an anaerobic environment, indicating that pyruvate
alone is insufficient to counteract all the inhibitory effects of
oxygen.
Recent research has established that growth-arrested,
stressed bacterial cells may undergo a process of respir-
ation-induced self-destruction when subsequently cultured
under aerobic conditions (Dodd et al. 1997; Aldsworth
et al. 1999), where respiratory metabolism produces a burst
of intracellular-free radicals that is uncoupled from growth.
For example, the transfer of starved cells to a nutrient-rich
medium can lead to the rapid production of ROS,
including superoxide and peroxides (Bloomfield et al.1998), and these may overwhelm the antioxidant defence
systems of such cells, resulting in their death. Given that
bacteria such as E. coli are normal inhabitants of the colon,
an anaerobic environment, it is possible that the conven-
tional (aerobic) approach to culture represents an artificial
situation under which such cells are not enumerated. One
means of preventing such self-destruction is to culture the
organisms in the complete absence of oxygen, forcing them
to use anaerobic pathways for energy metabolism. Stephens
et al. (2000) have shown that such an approach can be
extremely effective in culturing sublethally injured
Salmonella following exposure to heat, acid or salt stress,
demonstrating that the combined effects of (i) neutraliza-
tion of extracellular ROS generated in the growth medium
together with (ii) the prevention of intracellular (respirat-
ory) ROS consistently gave the highest counts. This is in
agreement with the findings of George and Peck (1998),
who have shown that E. coli cells injured by heat treatment
are subsequently unable to grow in a medium of high redox
potential, implicating the reactive by-products of oxidative
respiratory metabolism as the principal toxic agents respon-
sible for preventing the growth of heat-damaged cells
under aerobic conditions. The present study shows that a
similar enhancement of the count of UVA-treated E. coli,can be observed under ROS-neutralized enumeration
Table 2 Comparative inactivation of
Escherichia coli grown and illuminated in
water under different levels of oxygen. T90
values for E. coli NCTC8912 cultured under
air-sparged or N2-sparged conditions, and
then exposed to simulated sunlight for 6 h
under different levels of dissolved oxygen and
enumerated either (i) on nutrient agar under
aerobic conditions or (ii) on nutrient agar plus
0Æ05% pyruvate under anaerobic conditions
Oxygen status during
illumination
T90 values (min) for cells grown
under air-sparged conditions
(oxygenated)
T90 values (min) for cells grown
under N2-sparged conditions
(hypo-oxygenated)
Aerobic
Anaerobic + pyruvate
(% increase) Aerobic
Anaerobic + pyruvate
(% increase)
Hypo-oxygenated 63 105 (67) 247 424 (72)
Oxygenated 36 49 (36) 79 107 (35)
Hyperoxygenated 27 37 (37) 46 60 (30)
The percentage increase in T90 observed under anaerobic, pyruvate-supplemented conditions is
shown in brackets for each experimental treatment.
46 R. KHAENGRAENG AND R.H. REED
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
conditions, achieved by either (i) a combination of added
pyruvate with incubation in an anaerobic jar or (ii) prere-
duced growth medium maintained in an anaerobic cabinet.
Overall, the data for pyruvate supplementation indicate
that peroxide-based ROS are likely to account for the
predominant component of this inhibitory effect. The fact
that a positive effect of pyruvate supplementation was only
observed on aerobic plates transferred to an anaerobic
environment and not with prereduced plates maintained
under anaerobic conditions (Fig. 3) suggest that pyruvate
may protect against peroxide-based respiratory by-products
formed in the first hour or so after transfer to anaerobic
conditions from air, before the atmosphere and growth
medium are fully anaerobic.
The present study has demonstrated that a substantial
proportion of E. coli cells subjected to either UVA or
simulated sunlight become sublethally damaged, reaching a
point where they can no longer form colonies in air on an
agar-based medium. Following the approach of Zaske et al.(1980), the relative percentage of such injured cells can be
calculated by comparing the aerobic plate count for
unsupplemented medium (uninjured cells) with the equiv-
alent count for anaerobic, pyruvate-supplemented medium
(injured plus uninjured cells). On this basis, c. 99% of the
cells of E. coli NCTC 8912 subjected to simulated sunlight
for a period of 6 h were sublethally damaged, being capable
of forming colonies only under anaerobic, pyruvate-supple-
mented conditions and not under aerobic conditions
(Fig. 4). Such sublethally injured cells would be missed by
conventional aerobic counting procedures. Approximately
50% of these injured cells were able to grow aerobically in
the presence of pyruvate, which may indicate a lower level of
damage in such cells. However, pyruvate supplementation
alone was insufficient to provide a count of all of the viable
cells present under such conditions, and broadly similar
results were obtained with four other strains (Table 1),
including E. coli O157 NCTC12900.
The present research raises a question as to whether
studies on solar inactivation/disinfection may overestimate
the bactericidal effectiveness of sunlight when based solely
on enumeration by conventional aerobic plate counts, in
addition to the problems known to be caused by the use of
selective media (Block and Goswami 1995). The comparat-
ive significance of cells sublethally damaged by sunlight is
discussed in greater detail by Reed (2004), although it
should be noted also that Smith et al. (2000) have demon-
strated that sublethally damaged cells of Salmonellatyphimurium show a far lower infectivity to BALB/c mice,
and are therefore less likely to cause disease than their
undamaged counterparts.
While the current study focuses on the nonthermal effects
of UVA and solar illumination, there is often an additional
thermal component to solar disinfection under field condi-
tions, with a synergistic interaction between temperature
and light (e.g. Wegelin et al. 1994; McGuigan et al. 1998).However, the studies of George and Peck (1998) and
Bromberg et al. (1998) have demonstrated that heat-injured
cells also become oxygen sensitive during subsequent
culture. Based on such observations, together with the
current findings, we would suggest that researchers who
wish to establish the number of healthy and sublethally
damaged bacteria present in solar-treated natural waters
should consider enumeration under conditions where the
inhibitory effects of ROS-based self-destruction are avoided,
either using fully anaerobic conditions and a prereduced
growth medium, or using an anaerobic jar with a pyruvate-
supplemented medium, in order to evaluate the significance
of these findings under field conditions. A further factor in
natural waters will be the presence of exogenous com-
pounds, such as humic acids, that have the potential either
to act as photosensitizers, or to block the transmission of
inactivating solar radiation (e.g. Curtis et al. 1992).It is also worth noting that the present results may raise a
question regarding the measurement of T90 values for faecal
indicator bacteria based on conventional aerobic count data,
which is in widespread use for modelling the inactivation of
faecal bacteria in coastal water systems (e.g. Gameson and
Gould 1975; Guillaud et al. 1997). As Bloomfield et al.(1998) have pointed out, the lack of growth of sublethally
injured bacteria under aerobic conditions also provides an
alternative explanation for the so-called �viable noncultur-
able� hypothesis, as sublethally injured cells do not neces-
sarily enter a distinct physiological state, but are simply
unable to grow under conventional aerobic conditions, as a
result of self-generated ROS. It is tempting to speculate that
a count obtained under fully anaerobic conditions on a
nonselective medium under conditions where ROS are
neutralized or eliminated is likely to be more representative
of the true number of viable bacteria, as this major source of
growth inhibition has been removed.
The present study suggests that stationary phase cells
(Fig. 4) may be more resistant to solar illumination than
their log phase counterparts (c.f. data for oxygenated water
shown in Fig. 5), in agreement with earlier findings (Reed
1997). However, caution should be observed when compar-
ing these two sets of results, as the stationary phase cells
were grown without gas sparging whereas the log phase cells
were sparged with N2 during growth. A more important
point to note is that broadly similar overall responses were
observed for both stationary phase cells (Figs 1–4) and log
phase cells (Figs 5 and 6), with both types showing
enhanced counts under anaerobic conditions with added
pyruvate. This contrasts with the proposal that this
phenomenon should be most apparent in experiments using
exponentially growing cultures (Dodd et al. 1997), as suchcells will suffer the greatest imbalance between respiratory
PHOTOINACTIVATION OF E. COLI 47
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
metabolism and growth as a result of sublethal stress. The
findings of the current study may reflect the fact that the
damage caused by UVA and simulated sunlight is because of
photo-oxidation, which is likely to make such cells more
sensitive to subsequent oxidative stress, irrespective of their
growth phase. It is also worth noting that antioxidant
enzymes such as catalase may also be significantly damaged
during exposure to light (Kapuscinski and Mitchell 1981),
leading to an increased sensitivity to ROS. The enhanced
survival of log phase cells grown under aerobic conditions,
compared with those grown under anaerobic conditions,
following exposure to simulated sunlight (c.f. Figs 5 and 6)
also supports the idea that intracellular antioxidant defences
play a major role in determining the extent of sublethal
damage and death, as aerobically grown cells would be
expected to have a higher level of ROS-degrading enzymes,
e.g. catalase and superoxide dismutase.
ACKNOWLEDGEMENTS
Thanks are due to Dr K.G. McGuigan (Royal College of
Surgeons in Ireland, Dublin) for the loan of the solar
simulator, and to Dr J. Perry (Freeman Hospital, Newcastle
upon Tyne) for help with equipment (anaerobic jars
and anaerobic cabinet). Financial support was provided
by the Royal Thai Government and the Royal Society of
London.
REFERENCES
Acra, A., Karahagopian, Y., Raffoul, Z. and Dajani, R. (1980)
Disinfection of oral rehydration solutions by sunlight. Lancet ii,
1257–1258.
Acra, A., Raffoul, Z. and Karahagopian, Y. (1984) Solar Disinfection of
Drinking Water and Oral Rehydration Solutions: Guidelines for
Household Application in Developing Countries. New York: UNICEF.
Alam, M.Z.B. and Ohgaki, S. (2002) Role of hydrogen peroxide and
hydroxyl radicals in producing the residual effect of ultraviolet
radiation. Water Environ Res 74, 248–255.
Aldsworth, T.G., Sharman, R.L. and Dodd, C.E.R. (1999) Bacterial
suicide through stress. Cell Mol Life Sci 56, 378–383.
Alkan, U., Elliott, D.J. and Evison, L.M. (1995) Survival of enteric
bacteria in relation to simulated solar radiation and other environ-
mental factors in marine waters. Water Res 29, 2071–2081.
Anon (2000) Anaerocult� A Product Information Sheet. Darmstadt,
Germany: Merck VWR Diagnostics.
Anon (2001)World Water Day 2001: Water for Health – Taking Charge.
URL http://www.worldwaterday.org/2001/report/ch4.html, last
accessed 16 March 2005.
Anon (2002) The Microbiology of Drinking Water (2002) – Part 1 –
Water Quality and Public Health. Methods for the Examination of
Waters and Associated Materials. London, UK: Environment Agency.
URL http://www.dwi.gov.uk/regs/pdf/micro.htm, last accessed
16 March 2005.
Barcina, I., Gonzalez, J.M., Iriberri, J. and Egea, L. (1990) Survival
strategies of Escherichia coli and Enterococcus faecalis in illuminated
fresh and marine systems. J Appl Bacteriol 68, 189–198.
Block, S.S. and Goswami, D.Y. (1995) Chemically enhanced sunlight
for killing bacteria. J Solar Energy Eng 1, 431–437.
Bloomfield, S.F., Stewart, G.S.A.B., Dodd, C.E.R., Booth, I.R. and
Power, E.G.M. (1998) The viable but non-culturable phenomenon
explained? Microbiology 144, 1–3.
Bogosian, G., Aardema, N.D., Bourneuf, E.V., Morris, P.J.L. and
O’Neil, J.P. (2000) Recovery of hydrogen peroxide-sensitive cultur-
able cells of Vibrio vulnificus gives the appearance of resuscitation
from a viable but nonculturable state. J Bacteriol 182, 5070–5075.
Bromberg, R., George, S.M. and Peck, M.W. (1998) Oxygen
sensitivity of heated cells of Escherichia coli O157:H7. J Appl
Microbiol 85, 231–237.
Caslake, L.F., Connolly, D.J., Menon, V., Duncanson, C.M., Rojas, R.
and Tavakoli, J. (2004) Disinfection of contaminated water by using
solar irradiation. Appl Environ Microbiol 70, 1145–1150.
Clesceri, L.S., Eaton, A.D. and Greenberg, A.E. (1998) Standard
Methods for the Examination of Water and Wastewater, 20th edn.
Washington, DC: American Public Health Organisation.
Curtis, T.P., Mara, D.D. and Silva, S.A. (1992) Influence of pH,
oxygen and humic substances on ability of sunlight to damage fecal
coliforms in waste stabilization pond water. Appl Environ Microbiol
58, 1335–1343.
Czechowitz, S.M., Santos, O. and Zottola, E.A. (1996) Recovery of
thermally-stressed Escherichia coli O157:H7 by media supplemented
with pyruvate. Int J Food Microbiol 33, 275–284.
Davies, C.M. and Evison, L.M. (1991) Sunlight and the survival of
enteric bacteria in natural waters. J Appl Bacteriol 70, 265–274.
Davies-Colley, R.J., Donnison, A.M., Speed, D.J., Ross, C.M. and
Nagels, J.W. (1999) Inactivation of faecal indicator micro-organisms
in waste stabilisation ponds: interactions of environmental factors
with sunlight. Water Res 5, 1220–1230.
Dodd, C.E.R., Sharman, R.L., Bloomfield, S.F., Booth, I.R. and
Stewart, G.S.A.B. (1997) Inimical processes: bacterial self-destruc-
tion and sub-lethal injury. Trends Food Sci Technol 8, 238–241.
Downes, A. and Blunt, T.P. (1877) Researches on the effects of light
upon Bacteria and other organisms. Proc R Soc 28, 488–501.
Elstner, E. and Heupel, A. (1976) Formation of hydrogen peroxide by
isolated cell walls from horseradish, Armoracia lapathifolia Gilib.
Planta 130, 175–180.
Gameson, A.H.L. and Gould, D.J. (1975) Effects of solar radiation on
the mortality of some terrestrial bacteria in sea water. In Discharge of
Sewage from Sea Outfalls ed. Gameson, A.H.L. pp. 209–217. Oxford,
UK: Pergamon Press.
George, S.M. and Peck, M.W. (1998) Redox potential affects the
measured heat resistance of Escherichia coli O157:H7 independently
of oxygen concentration. Lett Appl Microbiol 27, 313–317.
George, S.M., Richardson, L.C.C., Pol, I.E. and Peck, M.W. (1998)
Effects of oxygen concentration and redox potential on recovery of
sublethally heat-damaged cells of Escherichia coli O157:H7, Salmon-
ella enteritidis and Listeria monocytogenes. J Appl Microbiol 84,
903–909.
Gnanou Besse, N. (2002) Influence of various environmental param-
eters and of detection procedures on the recovery of stressed Listeria
monocytogenes: a review. Food Microbiol 19, 221–234.
48 R. KHAENGRAENG AND R.H. REED
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
Gourmelon, M., Cillard, J. and Pommepuy, M. (1994) Visible light
damage to Escherichia coli in seawater – oxidative stress hypothesis.
J Appl Bacteriol 77, 105–112.
Guillaud, J.F., Derrien, A., Gourmelon, M. and Pommepuy, M. (1997)
T90 as a tool for engineers: interest and limits. Water Sci Technol 11,
277–281.
Joyce, T.M., McGuigan, K.G., Elmore-Meegan, M. and Conroy,
R.M. (1996) Inactivation of fecal bacteria in drinking water by solar
heating. Appl Environ Microbiol 62, 399–402.
Kapuscinski, R.B. and Mitchell, R. (1981) Solar radiation induces
sublethal injury in Escherichia coli in seawater. Appl Environ
Microbiol 41, 670–674.
Kehoe, S.C. (2001) Batch process solar disinfection of drinking water:
process and pathogenicity. PhD Thesis, Royal College of Surgeons in
Ireland, Dublin, Ireland.
Khaengraeng, R. (2004) Characterisation of solar photo-oxidative
disinfection. PhD Thesis, University of Northumbria, Newcastle
upon Tyne, UK.
Knabel, S.J. and Thielen, S.A. (1995) Enhanced recovery of severely
heat-injured thermotolerant Listeria monocytogenes from USDA and
FDA primary enrichment media using a novel, simple, strictly
anaerobic method. J Food Prot 58, 29–34.
Lawand, T.A., Ayoub, J. and Gichenje, H. (1997) Solar disinfection of
water using transparent plastic bags. RERIC Int Energy J 19, 37–44.
Mackey, B.M. and Seymour, D.A. (1987) The effect of catalase on
recovery of heat-injured DNA-repair mutants of Escherichia coli.
J Gen Microbiol 133, 1601–1610.
McDonald, L.C., Hackney, C.R. and Ray, B. (1983) Enhanced
recovery of injured Escherichia coli by compounds that degrade
hydrogen peroxide or block its formation. Appl Environ Microbiol 45,
360–365.
McFeters, G.A., Kippin, J.S. and LeChevallier, M.W. (1986) Injured
coliforms in drinking water. Appl Environ Microbiol 51, 1–5.
McGuigan, K.G., Joyce, T.M., Conroy, R.M., Gillespie, J.B. and
Elmore-Meegan, M. (1998) Solar disinfection of drinking water
contained in transparent plastic bottles: characterizing the bacterial
inactivation process. J Appl Microbiol 84, 1138–1148.
McGuigan, K.G., Joyce, T.M., Conroy, R.M. (1999) Solar disinfec-
tion: use of sunlight to decontaminate drinking water in developing
countries. J Med Microbiol 48, 785–787.
Meyer, V. and Reed, R.H. (2001) SOLAIR disinfection of coliform
bacteria in hand-drawn drinking water. Water SA 27, 49–52.
Mizunoe, Y., Wai, S.N., Takade, A. and Yoshida, S. (1999)
Restoration of culturability of starvation-stressed and low-tempera-
ture-stressed Escherichia coli O157 cells by using H2O2-degrading
compounds. Arch Microbiol 172, 63–67.
Mongkolsuk, S. and Helmann, J.D. (2002) Regulation of inducible
peroxide stress responses. Mol Microbiol 45, 9–15.
Murano, E.A. and Pierson, M.D. (1992) Effect of heat shock and
growth atmosphere on the heat resistance of Escherichia coli
O157:H7. J Food Prot 55, 171–175.
Nebra, Y., Jofre, J. and Blanch, A.R. (2002) The effect of reducing
agents on the recovery of injured Bifidobacterium cells. J Microbiol
Methods 49, 247–254.
Oufdou,K.,Mezrioui,N.,Oudra,B.,Barakate,M.,Loudiki,M. andAlla,
A.A. (2000) Relationships between bacteria and cyanobacteria in the
Marrakech waste stabilisation ponds.Water Sci Technol 42, 171–178.
Potters, G., De Gara, L., Asard, H. and Horemans, N. (2002)
Ascorbate and glutathione: guardians of the cell cycle, partners in
crime? Plant Physiol Biochem 40, 537–548.
Reasoner, D.J. and Geldrich, E.E. (1985) A new medium for the
enumeration and subculture of bacteria from potable water.
Appl Environ Microbiol 49, 1–7.
Reed, R.H. (1996) Sol-air water treatment. In Proceedings of the
Twenty-second Water, Engineering and Development Centre Conference,
New Delhi, India ed. Pickford, J. pp. 259–260. Loughborough, UK:
University of Loughborough.
Reed, R.H. (1997) Solar inactivation of faecal bacteria in water: the
critical role of oxygen. Lett Appl Microbiol 24, 276–280.
Reed, R.H. (2004) The inactivation of microbes by sunlight: solar
disinfection as a water treatment process. Adv Appl Microbiol 54,
333–365.
Reed, R.H., Mani, S.K. and Meyer, V. (2000) Solar photo-oxidative
disinfection of drinking water: preliminary field observations.
Lett Appl Microbiol 30, 432–436.
Runyen-Janecky, L.J., Hong, M. and Payne, S.M. (1999) The
virulence plasmid-encoded impCAB operon enhances survival and
induced mutagenesis in Shigella flexneri after exposure to UV
radiation. Infect Immun 67, 1415–1423.
Sartory, D.P. (1995) Improved recovery of chlorine-stressed coliforms
with pyruvate-supplemented media. Water Sci Technol 31, 255–258.
Sartory, D.P. and Howard, L. (1992) A medium detecting
b-glucuronidase for the simultaneous membrane filtration enumer-
ation of Escherichia coli and coliforms from drinking water. Lett Appl
Microbiol 15, 273–276.
Schoeller, N.P. and Ingham, S.C. (2003) Comparison of the Baird-
Parker agar and 3 MTM PetrifilmTM rapid S. aureus plate count
methods for detection and enumeration of Staphylococcus aureus.
Food Microbiol 18, 581–587.
Shen, B., Jensen, R.G. and Bohnert, H.J. (1997) Mannitol protects
against oxidation by hydroxyl radicals. Plant Physiol 115, 527–532.
Singh, D.P. andKshatriya, K. (2002) NaCl-induced oxidative damage in
the cyanobacterium, Anabaena doliolum. Curr Microbiol 44, 411–417.
Sinton, L.W., Finlay, R.K. and Lynch, P.A. (1999) Sunlight
inactivation of fecal bacteriophages and bacteria in sewage-polluted
seawater. Appl Environ Microbiol 65, 3605–3613.
Sinton, L.W., Hall, C.H., Lynch, P.A. and Davies-Colley, R.J. (2002)
Sunlight inactivation of fecal indicator bacteria and bacteriophages
from waste stabilisation pond effluent in fresh and saline waters.
Appl Environ Microbiol 68, 1122–1131.
Smith, R.J., Kehoe, S.C., McGuigan, K.J. and Barer, M.J. (2000)
Effects of simulated solar disinfection of water on infectivity of
Salmonella typhimurium. Lett Appl Microbiol 31, 284–288.
Sommer, B., Marino, A., Solarte, Y., Salas, M.L., Dierolf, C.,
Valiente, C., Mora, D., Rechsteiner, R. et al. (1997) SODIS – an
emerging water treatment process. J Water Sci Res Tech – Aqua 46,
127–137.
Stephens, P.J., Druggan, P. and Caron, N-V. (2000) Stressed
Salmonella are exposed to reactive oxygen species from two
independent sources during recovery in conventional culture media.
Int J Food Microbiol 60, 269–285.
Suh, J.H. and Knabel, S.J. (2000) Comparison of different reducing
agents for enhanced detection of heat-injured Listeria monocytogenes.
J Food Prot 63, 1058–1063.
PHOTOINACTIVATION OF E. COLI 49
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x
Voelker, B.M., Morel, F.M.M. and Sulzberger, B. (1997) Iron redox
cycling in surface waters: effects of humic substances and light.
Environ Sci Technol 31, 1004–1011.
Walker, D.C., Len, S-V. and Sheehan, B. (2004) Development and
evaluation of a reflective solar disinfection pouch for treatment of
drinking water. Appl Environ Microbiol 70, 2545–2550.
Wegelin, M., Canonica, S., Mechsner, K., Fleischmann, T., Pesaro, F.
and Metzler, A. (1994) Solar water disinfection: scope of the process
and analysis of radiation experiments. J Water Sci Res Tech – Aqua
43, 154–169.
Xavier, I.J. and Ingham, S. (1993) Increased D-values for Salmonella
enteritidis resulting from the use of anaerobic enumeration methods.
Food Microbiol 10, 223–228.
Zaske, S.K., Dockins, W.S., Schillinger, J.F. and McFeters, G.A.
(1980) New methods to assess bacterial injury in water. Appl Environ
Microbiol 39, 656–658.
50 R. KHAENGRAENG AND R.H. REED
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x