APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p. 1170-1176
Copyright C 1977American Society for Microbiology
Vol. 33, No. 5
Printed in U.S.A.
Effect of Combined Heat and Radiation on Microbial
D. A. FISHER2 AND I. J. PFLUG*
Department ofFood Science and Nutirition and the School ofPublic Health, University ofMinnesota,
St. Paul, Minnesota 55108
Received for publication 22 October 1976
A series of experiments at several levels of relative humidity and radiation
dose rates was carried out using spores ofBacillus subtilis var. niger to evaluate
the effect of heat alone, radiation alone, and a combination of heat and radia-
tion. Combined heat and radiation treatment of microorganisms yields a de-
struction rate greater than the additive rates of the independent agents. The
synergistic mechanism shows a proportional dependency on radiation dose rate,
an Arrhenius dependency on temperature, and a dependency on relative humid-
ity. Maximum synergism occurs under conditions where heat and radiation
individually destroy microorganisms at approximately equal rates. Larger syn-
ergistic advantage is possible at low relative humidities rather than at high
One of the more intriguing subjects of the
Planetary Quarantine research has been the
discovery of the synergistic effect that results
from the combination of heat and radiation for
bacterial spore destruction. Koesterer (2) ob-
served this effect while carrying out explora-
tory sterilization studies for the National Aeu-
ronautics and Space Administration (NASA).
Scientists at the Sandia Laboratories carried
out extensive laboratory and feasibility studies
on the use of combined heat and radiation for
spacecraft sterilization (5).
This report describes studies carried out at
the University of Minnesota to investigate the
sterilization attributes of the thermoradiation
process. Destruction rate tests were carried out
at a number of radiation levels, temperatures,
and relative humidities. Both wet- and dry-
heat conditions were used. We have attempted
not only to develop destruction rate data but
also to determine the mechanistic basis for the
synergism displayed by these seemingly inde-
pendent lethal agents.
MATERIALS AND METHODS
Biological procedures. Bacillus subtilis var. niger
spores grown at 32°C in synthetic sporulation me-
dium-10 (3) were used in this study. The spores were
cleaned by exposure to ultrasonic energy and by
repeated washing with deionized distilled water and
'Minnesota Agricultural Experiment Station Scientific
Journal Series paper no. 9800.
2Present address: E. I. Dupont de Nemours, Richmond,
For the wet-heat studies, the spores were sus-
pended in 5 ml ofSorensen 0.067 M phosphate buffer
(pH 7.0) in screw-capped glass test tubes. The popu-
lation density of the suspension was approximately
106 spores per ml. After inoculation the tubes were
refrigerated at 40C until treated. After treatment
the tubes were placed in an ice bath until assayed.
In the assay procedure, the sample was mixed and a
1-ml portion was diluted in buffered distilled water
(described in references 4), and duplicate portions
were plated using Trypticase soy agar (BBL).
For the dry-heat studies, a 0.01-ml portion of an
ethanol suspension of the spores was deposited on
stainless steel planchets (12.7 by 12.7 mm, 106
spores per planchet). The planchets were then equil-
ibrated at 22°C, 50% relative humidity, for at least
24 h before treatment. Samples were moved as
needed to the University ofMinnesota Gamma Irra-
diation Facility for testing. After treatment, the
planchets were placed in ice-cooled flasks until as-
sayed using NASA Standard Procedures (4). In the
assay procedure, buffered distilled water was added
to each flask, the flask was suspended in an ultra-
sonic (25 kHz, 0.35 W/cm2) tank filled with an
aqueous solution containing 0.3% Tween 80 (de-
scribed in reference 4) for 2 min, and duplicate por-
tions ofthe eluate were plated using Trypticase soy
All inoculation and recovery procedures were car-
ried out in a class 100 clean room. Colony-forming
units were counted after 48 h of incubation at 32°C.
Radiation system. The University of Minnesota
Gamma Irradiation Facility uses a cylindrical array
of cesium-137 sources of approximately 10,000 Ci.
The radiation field was mapped using Fricke Dosi-
metry as a primary reference and a calibrated Victo-
reen rate meter as a secondary reference.
Before biological testing, areas within the radia-
EFFECT OF COMBINED HEAT AND RADIATION
tion field with the desired intensities
fied. When all of the environmental s
ment was in place in the radiation fac
locations were retested using Fricke dc
ples in the environmental chambers.
these tests indicated that radiation int
not significantly affected either by ab
environmental chamber walls or bar
from support equipment. Exposure doi
10, and 5 krd/h were used.
Heating systems. For wet-heat expei
ples sealed in test tubes were placed ih
chamber of the type shown in Fig. 1.
FIG. 1. Sectional view of exposure
in wet-heat thermoradiation study.
3 were identi-
The results of
se rates of 20,
was a tubular thin-walled aluminum vessel through
which water was circulated from a constant temper-
ature bath. The system heated the sample within
1°C of bath temperature within 50 s after start-up.
This time lag was short relative to treatment times
used in the experiment.
For dry-heat experiments, the inoculated plan-
chets were clamped in an environmental chamber at
positions of known radiation intensities. Tempera-
ture control was provided through heaters located
along the base of the chamber. The humidity of the
air was controlled by controlling the dew point ofthe
air circulating through the chamber; a diagram of
the environmental control system is shown in Fig. 2.
Thestart-upand endproceduresofeach test were
selected to apply or remove both biological stress
factors simultaneously. The start or end of the ra-
diation treatment was determined by the position of
the radiation source elevator. The end point of the
thermal treatments was accomplished by making
step changes in the thermal stress environment. For
wet-heat experimentation, start was taken as the
time when circulation of the heated water was
started. The samples were quenched in cool water at
the end of the test. For dry-heat experiments, start
was the time when the humidity conditions were
changed rapidly producinga step change from non-
lethal to lethal conditions. At the end of the test a
reverse change in humidity was made.
throughout the tests using thermocouples located at
each sample site; they were recorded using a tem-
conditions were monitored using wet- and dry-bulb
thermocouples located in a chamber at the rear of
the environmental chamber.
Several spore samples were treated simultane-
ously. Treatment periods were successive to one an-
n an exposure
WET AND DRY
FIG. 2. Schematic flow chart forthe environmental system in thermoradiation experiments.
VOL. 33, 1977
FISHER AND PFLUG
(7% RH, U Kruinrr
FIG. 3. Survivor curves for dry radiation, dry-heat thermal, and thermoradiation ofB. subtilis var. niger
(AAHK) at 110°C, 27% relative humidity, and 20 krd/h.
TABLE 1. D-values (hours) at various temperatures
and exposure rates for wet-heat thermoradiation of
Bacillus subtillis var. niger (AAHF)
a Dose rates (krd/h).
bEssentially infinite for
cEstimate (insufficient data for accurate analy-
time span of experi-
other, and a randomized treatment sequence was
Semilogarithmic survivor curves were con-
structed from the data gathered at each test
TABLE 2. D-values at various temperatures and
exposure rates for dry-heat thermoradiation of
Bacillus subtilis var. niger (AAHK)
a Geometric mean of two to six D-value determinations
from separate experiments.
b Dose rates (krd/h).
condition. The survivor curves for one set of
thermoradiation conditions are shown in Fig. 3;
the synergistic effect is illustrated by this
graph. The destruction rate obtained using the
(27°C, 50% RH, 20 Kr(
::.-- -_-Z-i -.----+---
APPL. ENVIRON. MICROBIOL.
EFFECT OF COMBINED HEAT AND RADIATION
simultaneous application ofheat and radiation
is greater than the additive microbial destruc-
tion rate of the individual agents.
The D-values obtained from wet- and dry-
heat thermoradiation experiments are shown
in Tables 1 and 2. The D-value is the reciprocal
ofthe slope ofthe straight-line semilogarithmic
survivor curve; it is measured as the treatment
time necessary to reduce the number of spores
by 90% and is calculated from the results ofthe
linear regression analysis of spore survivor
data. DT, DR, and DTR are specific D-values for
temperature, radiation, and combined temper-
ature and radiation.
It is convenient to quantify the synergism in
terms of a synergism index (SI):
SI = thermoradiation destruction rate/
thermal destruction rate
+ radiation destruction rate
The SI as a function of the relative rates for
the thermal and radiation destruction tests for
FIG. 4. Values ofSI calculated from experimental wet-heat thermoradiation study.
DT I DR
FIG. 5. Values ofSI calculated from experimental dry-heat thermoradiation study.
VOL. 33, 1977
FISHER AND PFLUG
100 9080 70 60 50 403020
FIG.6. Arrhenius plot of the D-value (hours) for
radiation-induced deathfor wet-heat thermoradia-
wet heat are shown in Fig. 4 and for dry heat in
Fig. 5. (D1R,
is the D-value obtained from radia-
tion tests carried out at ambient temperatures.)
Insightsinto the thermoradiation
nism can be gained by examining the relative
experimental response as temperature,
tion dose rate,and psychrometric conditions
Radiation-induced microbial destruction ki-
netics are expected to have a temperature de-
pendency of the Arrhenius form: DRt = h&'^
where k = preexponential coefficient, e = Na-
perian base (2.71828. . .), E4 = activation en-
ergy, R = gas constant, and T = absolute tem-
perature. The measured energy of activation
was 110 cal/mol over the range -143 to 36°C in
Bacillus megaterium (6). An Arrhenius plot of
D-values for radiation-induced destruction (DR)
from wet-heat thermoradiation experiments is
shown in Fig. 6. The wet-heat contribution to
microbial destruction has been subtracted us-
ing the following equation:
D.,; where DR¢ = D-value for radiation-induced
= D-value from thermoradia-
tion experiments, and D1. = D-value from ther-
1/DR =i/DiR - 1/
mal experiment performed at same tempera-
ture as thermoradiation experiment. Plotted in
this form, the graph would be a straight line if
only one mechanism were present with an Ar-
rhenius temperature dependency. In reality,
the curves remain approximately horizontal
over the lower range oftemperatures but break
sharply downward over the higher temperature
range. This behavior suggests that different
mechanisms are dominant over different tem-
perature ranges. At high temperatures, the
synergistic mechanism becomes effective.
Wet-heat starts to be an effective steriliza-
tion tool at about 75°C. The D-value at that
temperature is approximately 67 h. This is the
temperature where the synergistic effect also
becomes noticeable. When we examined all of
our data where synergism was apparent, heat
by itself was a lethal agent.
Curves in Fig. 6 corresponding to different
radiation levels are similar in shape. Over the
range oftemperatures studied, the vertical dis-
tance between any two adjacent curves remains
approximately equal to log 2 (0.301). Thus the
preexponential coefficient of the kinetic "con-
stant" is proportional to radiation dose rate.
The energy of activation calculated over the
temperature range of 75 to 90°C for radiation-
induced spore destruction in 27.3 kcal/mol (ca.
114.3 KJ/mol) compared with 65.3 kcal/mol (ca.
273.4 KJ/mol) for thermal destruction over the
same temperature range.
In dry-heat thermoradiation experiments,
the amount of synergism depends on the rela-
tive humidity. A graph of D-values for radia-
tion-induced destruction from dry-heat experi-
ments as a function of relative humidity is
shown in Fig. 7. Once again, the thermal con-
tribution has been uncoupled from the total
destruction rate. The resulting D-value for DR
shows a dependency on relative humidity. This
dependency is of the same form as isothermal
D-value data for thermally induced mecha-
nisms (Fig. 8), although it is somewhat weaker.
Vertical distance between curves for a 2x dose
rate difference is approximately equal to log 2
Comparison ofDR withDR, shows that DR is
consistently less than DRO. The differences be-
tween DR andDR, represent the destruction
caused by the synergism mechanism.
The location ofthe maximum point for syner-
gism is vital in order to take the maximum
advantage ofthermoradiation as a sterilization
method. Optimum advantage is gained when SI
is maximized; i.e., thermoradiation death rate
is maximized relative to the additive rates of
heat and radiation. As seen in Fig. 4 and 5,
APPL. ENVIRON. MICROBIOL.