Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Destruction of Bacillus licheniformis spores by microwave

irradiation
S.-Y. Kim1,2, S.J. Shin1,2, C.-H. Song1, E.-K. Jo1,2, H.-J. Kim1 and J.-K. Park1,3
1 Department of Microbiology, College of Medicine, Chungnam National University, Daejeon, Korea
2 Infectious Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon, Korea
3 Cancer Research Institute, College of Medicine, Chungnam National University, Daejeon, Korea

Keywords
Bacillus licheniformis, microwave irradiation,
spore, spore killing, sporicidal mechanism.
Correspondence
Jeong-Kyu Park, Department of Microbiology,
College of Medicine, Chungnam National
University, Daejeon 301-747, Korea.
E-mail: jekpark@cnu.ac.kr
2007 ⁄ 1743: received 31 October 2007,
revised 5 August 2008 and accepted
1 September 2008
doi:10.1111/j.1365-2672.2008.04056.x
Abstract
Aims: To investigate the sporicidal mechanisms of microwave irradiation on
Bacillus licheniformis spores.
Methods and Results: We measured spore viability and the release of DNA
and proteins, and performed transmission electron microscopy (TEM).
A microwave oven (0Æ5 kW) was modified to output power at 2Æ0 kW, which
allowed a shorter sterilization cycle. A 2Æ0 kW microwave treatment at the
boiling temperature for 1 min did not kill all spores, but killed most spores.
The spore inactivation rate was faster than that of boiling and 0Æ5 kW micro-
wave oven. In contrast to boiling and 0Æ5 kW microwave treatments, the
2Æ0 kW microwave resulted in significant leakage of proteins and DNA from
spores due to injury to the spore structure. TEM revealed that 2Æ0 kW micro-
wave irradiation affected spore cortex hydrolysis and swelling, and ruptured
the spore coat and inner membrane.
Conclusions: These results suggest that 2Æ0 kW microwave irradiation ruptures
the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study: This study provides information on the
sporicidal mechanisms of microwave irradiation on B. licheniformis spores.

ting cell membrane damage. Structural alterations to the

Introduction
Microwaves are used for many purposes, such as steriliz-
ing hospital waste, decomposing organic matter, killing
pathogens in food and sterilizing medical utensils. The
heating mechanism, microbicidal mechanism and indus-
trial applications of microwaves have been studied (Lati-
mer and Matsen 1977; Vaid and Bishop 1998; Sudrik
et al. 2002; Celandroni et al. 2004; Elhafi et al. 2004;
Hong et al. 2004).
Although there is conflicting evidence regarding
whether microbial inactivation is due to heating or to the
electromagnetic energy itself, microwave and boiling
treatment are known to have different bactericidal mecha-
nisms (Wu 1996; Yeo et al. 1999; Sudrik et al. 2002).
Exposing nonspore-forming bacterial suspensions to
microwave irradiation causes reduced viable cell counts
and increases the leaching of DNA and proteins, sugges-
cell wall of Escherichia coli and fecal coliform bacteria
have been observed after microwave irradiation, but
exposure to boiling does not lead to structural alterations
(Woo et al. 2000; Hong et al. 2004). Therefore, the bacte-
ricidal mechanisms of microwave irradiation are involved
in cell membrane and cell wall damage, leading to protein
and DNA leakage.
Compared with the number of investigations on the
bactericidal mechanisms of microwave irradiation, there
have been few studies on the sporicidal mechanisms of
microwaves. It has been shown previously that microwave
irradiation of Clostridium sporogenes disrupts spores and
causes the release of DNA and calcium ions from the
spore core, but exposure to boiling does not. It is unclear
why spores fragment under these conditions, but it might
be the result of increased internal pressure within the
core. Conventional methods for destroying cells often rely

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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885 877
Spore killing mechanisms by microwave irradiation
on the generation of external pressure (Vaid and Bishop
1998). An analysis of dipicolinic acid (DPA) release and
electron microscopy of treated spores revealed that spore
damage induced by microwaves is significantly different
from the boiling. DPA is detectable in the supernatant of
heated spore suspensions, whereas no DPA is found after
spore irradiation (Celandroni et al. 2004). Our results
demonstrate that the dramatic effects of microwave expo-
sure on the ultrastructure of bacterial spores involve a
mechanism different from that of conventional heating
such as boiling or autoclaving.
Although the microwave power level has no significant
effect on E. coli destruction (Yaghmaee and Durance
2005), less time is required to reach the boiling point
with a high output power (750 W) than with a low
power level (80 W) (Celandroni et al. 2004). Therefore,
we modified a microwave oven (Model RE-500W; Sam-
sung, Inc., Suwoon, Korea) to provide a power output of
2Æ0 kW, thus allowing a shorter sterilization cycle.
Bacillus subtilis is commonly used to determine the
effectiveness of sterilization procedures in hospital and
industry settings (Melly et al. 2002) and is considered an
optimum indicator bacterium for microwave sterilization.
Bacillus licheniformis is a spore-forming organism. The
spores of different Bacillus species (B. licheniformis, Bacil-
lus cereus, Bacillus coagulans and B. subtilis) differ in their
resistance to microwave irradiation, with B. licheniformis
spores having the highest resistance (Wang et al. 2003).
The sporicidal mechanisms of microwave treatment
remain unknown. In this study, the utility and mecha-
nism of microwave sporicidal activity were studied. The
destruction of bacterial endospores by microwave irradia-
tion was investigated by performing bacterial viability and
laccase activity assays, measuring the release of DNA and
proteins, and examining transmission electron micro-
graphs.
Materials and methods
Isolation of B. licheniformis from Chung-kook-jang
A B. licheniformis strain was isolated from Korean
Chung-kook-jang, a traditional Korean fermented soybean
food made of micro-organisms in rice straw and boiled
soybean. Chung-kook-jang was diluted 1 : 10 in buffered
peptone-water (Oxoid) and resuspended by vigorous mix-
ing on a vortex until an evenly distributed suspension
was obtained. The suspension was incubated for 24 h at
37C. Subsequently, 0Æ1 ml aliquots were plated aerobi-
cally on nutrient agar (Difco Laboratories, Detroit, MI,
USA) and incubated for 72 h. Colonies with different
morphologies were picked at random and purified by
streaking on agar plates of the same medium. The spores
878 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885
S.-Y. Kim et al.
were confirmed with Schaeffer–Fulton stain (Hamouda
et al. 2002). The taxonomical position of this strain was
determined using a Microgen Bacillus-ID (Microgen
Bioproducts Ltd., Surrey, UK). This strain was stored at
)80C in nutrient broth containing 20% (v ⁄ v) glycerol.
These stock cultures were used as starting material for the
following experiments.
Spore preparation
For induction of spore formation, B. licheniformis colo-
nies were grown on nutrient agar at 37C for 1 week.
Spores and sporulating cells were scraped from the plates,
and the spores were purified as described previously
(Paidhungat et al. 2000), with slight modifications. The
bacteria and spores were suspended in lysis buffer
(0Æ015 mol l)1 NaOH, 0Æ5 mol l)1 NaCl, 0Æ05% N-lauryl-
sarcosinate, pH 12Æ5) and incubated on ice for 3 h, to lyse
the remaining vegetative bacteria. The suspension was
centrifuged at 200 g (Allegra 6R; Beckman Coulter, Full-
erton, CA, USA) for 20 min, and the pellet was washed
in cold deionized water. The suspension was centrifuged
at 1200 g for 20 min, and the pellet was resuspended in
0Æ1 mol l)1 phosphate buffer (PB, pH 7Æ2). Any nonlysed
bacteria were removed by filtration through a 0Æ45 lm
membrane (Satorius; Weender Landstrasse, Goettingen,
Germany). The prepared spores were confirmed by Scha-
effer–Fulton staining (Hamouda et al. 2002). The spore
suspension was smeared on a glass slide and heat fixed.
The slides were flooded with 5% aqueous malachite green
(Fisher Scientific Co., Fair lawn, NJ, USA) and intermit-
tently heated with a flame for 5 min to ensure that the
dye remained hot but not boiling. The slides were rinsed
with tap water and then soaked in 0Æ5% Safranin-O
(Sigma, St Louis, MO, USA) for 1 min. After drying, the
slides were examined using oil immersion and a light
microscope. All spore preparations used in this work were
free of growing or sporulating cells and germinated
spores.
Viability test of spores in suspension
Aliquots (2 ml) of suspension were microwave-irradiated
at 0Æ5 or 2Æ0 kW, or boiled, for 0, 0Æ5, 1, 2, 3 or 150 min.
Control samples (exposure to 0 time) contained spores
that did not undergo any treatment. For the microwave
heating, a household microwave oven (Model RE-500W,
2450 MHz; Samsung) and a larger-cavity oven, rated at
2Æ0 kW were used in this study. The updated oven was
offered from Energy Conversion & Storage Research Cen-
ter, Korea Institute of Energy Research (Korea).
A sterilized test-tube containing 2 ml of each spore sus-
pension to be heated in oven was placed in 150-ml Pyrex
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S.-Y. Kim et al.
120
100
Temperature (°C)
80
60
40
20
0 To investigate cell membrane damage caused by boiling
0 5 10 15 20 25 30 60 90 120 150 180
Time (s)
Figure 1 Changes in the temperature of suspensions relative to
microwave exposure time. A spore suspensions in 0Æ1 mol l)1 phos-
phate buffer was exposed to microwave irradiation at 0Æ5 kW (filled
circles) or 2Æ0 kW (open circles), and its temperature changes were
monitored.
beaker. The test-tube was placed together with the beaker
on the centre of the table and the microwave was irradia-
ted for various periods of times. For boiling, samples
were conventionally heated for the same time intervals by
immersion at 99C in a boiling water bath. The tempera-
ture of the spore suspension was determined with a digi-
tal thermometer (Model DT-615; CEM, Shenzhen, China)
attached a temperature probe. It was laid on the sample
tube in microwave oven and the temperature was
measured simultaneously during the irradiation.
Figure 1 shows the correlation between the microwave
radiation time and the temperature changes. To count the
surviving spores, a 100 ll aliquot of each treated suspen-
sion was serially diluted with PB and plated in triplicate
on nutrient agar. After the plates were incubated for 18 h
at 37C, the colony count was determined and expressed
as the average number of surviving cells (Yaghmaee and
Durance 2005). The number of colony forming units per
millilitre (CFU ml)1) was determined, and the number
was log10-transformed. All experiments were performed in
triplicate.
Viability test of spores on a filter membrane
The purified spores were suspended in PB and serially
diluted. The diluted suspension was filtered through a
0Æ22 lm membrane (diameter 47 mm, thickness 180 lm,
GSWP; Millipore, Bedford, MA, USA), and the
membranes were dried at 100-mm petridish with air
circulation in hood, until the membranes were changed
transparent to translucent. There are no quantitative data
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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885
Spore killing mechanisms by microwave irradiation
to support that membranes were indeed dry. The dried
membranes were microwave-irradiated at 0Æ5 or 2Æ0 kW
for 0, 5, 15, 30, 60, or 90 min. The treated membranes
were incubated on nutrient agar for 18 h at 37C, and
the colony count was determined and expressed as the
average number of surviving cells. The number of
CFU ml)1 was determined and log10-transformed. All
experiments were performed in triplicate.
Assay for spore structural damage by microwave
irradiation
and irradiation, we measured the release of intracellular
proteins and nucleic acids of B. licheniformis spore sus-
pensions (Vaid and Bishop 1998; Woo et al. 2000; Hong
et al. 2004). The spores were suspended in PB, and a
2 ml aliquot of suspension was microwave-irradiated at
0Æ5 or 2Æ0 kW, or boiled, for 0, 20, 40, or 60 s.
The amount of protein released from the microwaved
and the boiled suspensions was measured at 595 nm by
the Bradford (1976) method, with bovine serum albumin
as the standard protein. The nucleic acid content of the
supernatant was directly measured using a UV spectro-
photometer (Beckman DU 530) at 260 nm (Sambrook
and Russel 2001). The spore coat protein, CotA, of
B. licheniformis displays laccase activity (Johannes and
Majcherczyk 2000; Cantarella et al. 2003). Laccase activity
was routinely assayed at 37C using the 2,2¢-azinobis
(3-ethylbenzthiazoline-6-sulfonate) (ABTS; Sigma) sub-
strate. The assay mixture contained 1 mmol l)1 ABTS
and 0Æ1 mol l)1 citrate buffer (0Æ1 mol l)1 citric acid,
0Æ1 mol l)1 sodium citrate, pH 5Æ0). ABTS oxidation was
measured at 405 nm using a UV spectrophotometer. All
experiments were performed in triplicate.
Effects of microwave irradiation on the ultrastructure of
B. licheniformis
Spores were suspended in PB, and 2 ml aliquots of sus-
pension were microwave-irradiated at the boiling tempe-
rature for 1 min, boiled at the boiling temperature for
3 min, or autoclaved at 121C for 15 min. The treated
suspensions were centrifuged and fixed with 2Æ5% gluta-
raldehyde in PB for 2 h at 4C. The cells were washed
twice with 0Æ1 mol l)1 sodium phosphate buffer and then
postfixed with 1% osmium tetraoxide in PB for 1Æ5 h at
room temperature. The specimens were dehydrated in a
graded series of ethanol and embedded in Epon 812.
Ultrathin sections were obtained, contrasted with uranyl
acetate and lead citrate, and observed with a Hitachi
H-600 (Hitachi Co., Tokyo, Japan) transmission electron
microscope at an operating voltage of 75 kV.
879
Spore killing mechanisms by microwave irradiation S.-Y. Kim et al.

Table 1 The logarithmic reduction and

0Æ5 kW microwaved 2Æ0 kW microwaved Boiled survival of Bacillus licheniformis spores
Time Log Viability Log Viability Log Viability suspended in phosphate buffer treated with
(min) CFU ml)1 (%) CFU ml)1 (%) CFU ml)1 (%) microwaves and boiling

0 7Æ81 100 8Æ38 100 7Æ75 100

0Æ5 7Æ69 77Æ3 6Æ51 1Æ33 7Æ73 96Æ4
1 7Æ64 54Æ7 6Æ41 1Æ08 7Æ73 96Æ4
2 7Æ08 18Æ8 6Æ26 0Æ75 7Æ73 94Æ6
3 6Æ66 7Æ2 6Æ15 0Æ58 7Æ72 94Æ6
10 NA NA NA NA 7Æ71 91Æ1
30 NA NA NA NA 7Æ60 71Æ4
60 NA NA NA NA 7Æ43 48Æ2
90 NA NA NA NA 6Æ95 16Æ1
120 NA NA NA NA 5Æ85 1Æ3
150 NA NA NA NA – 0Æ0

NA, not applicable.

Table 2 The logarithmic reduction and survival of Bacillus lichenifor-

Results
mis spores on membrane filter treated with microwaves

Effect of microwave irradiation on the viability of 0Æ5 kW microwaved 2Æ0 kW microwaved

B. licheniformis spores
Time Log Viability Log Viability
The temperature of spore suspensions (2 ml) increased (min) CFU ml)1 (%) CFU ml)1 (%)
from 25C to the boiling point within 30 s in a 0Æ5 kW 0 7Æ40 100 7Æ40 100
microwave oven and within 20 s in a 2Æ0 kW microwave 5 6Æ20 6Æ4 5Æ85 2Æ8
oven (Fig. 1). Bacillus licheniformis spores suspended in 15 6Æ00 4Æ0 5Æ78 2Æ4
PB exhibited a 1-log reduction after 3 min of 0Æ5 kW 30 5Æ60 1Æ6 4Æ78 0Æ24
microwave irradiation and a 2-log reduction after 1 min 60 3Æ48 0Æ012 ND 0
of 2Æ0 kW microwave irradiation (Table 1). In contrast to 90 ND 0 ND 0

the microwave treatment, boiling did not lead to a signifi-
cant reduction in spore number after 3 min (7Æ75 vs
7Æ72 log CFU ml)1 in the control).
Boiling for 150 min was required for complete spore
inactivation (Table 1). However, we did not observe com-
plete sporicidal activity with our microwave irradiation
procedure because of evaporative losses from the spore
suspensions. Therefore, microbiological samples should be
in a dry state for microwave treatment. Spores were posi-
tively retained on the membrane filter surface, and the fil-
ters absorbed little of the fluid being filtered. Thus, the
filtering process was useful for determining the effect of
microwave irradiation on the viability of B. licheniformis
spores. Complete inactivation of B. licheniformis spores
was obtained with 90 min of microwave exposure at
0Æ5 kW and with 60 min at 2Æ0 kW (Table 2). These
results indicate that the spore inactivation rate was faster
with 2Æ0 kW microwave irradiation than with 0Æ5 kW
microwave irradiation or boiling.
Spore structural damage by microwave irradiation
With exposure to microwave irradiation, spores exhibited
a 0Æ17-log reduction after 1 min of 0Æ5 kW microwave
ND, Not detectable.
irradiation and a 1Æ97-log reduction after 1 min of
2Æ0-kW microwave irradiation (Table 1). Spore structural
damage caused by boiling and microwave irradiation for
1 min was investigated by measuring the release of intra-
cellular proteins and nucleic acids into the suspension
and the loss of laccase activity.
Spores exposed to 0Æ5- or 2Æ0 kW microwave irradia-
tion, or boiling, for 1 min released 5Æ5, 22Æ6, or
3Æ4 lg ll)1 of protein, respectively (Fig. 2). The protein
released from 0Æ5 kW microwave-irradiated spores was
twofold that released from untreated spores; the protein
released from 2-kW microwave-irradiated spores was 10-
fold that released from untreated spores. The amount of
protein released was significantly different between 0Æ5-
and 2Æ0-kW microwave-irradiated spores. There was no
difference in the amount of protein released between
boiled and untreated spores.
The amount of nucleic acid released from spores
exposed to 0Æ5- or 2Æ0 kW microwave irradiation, or boi-
ling, for 1 min was 10Æ5, 89Æ5 or 4Æ0 lg ll)1, respectively
(Fig. 3). The amount of nucleic acid released from

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880 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885
S.-Y. Kim et al.
25
Protein leaked (µg ml–1)
20
15
10
5 0·2
0 0·0
0 20 40
Time (s)
Figure 2 The amount of protein released into the spore suspension
of Bacillus licheniformis. The suspensions of isolated spores were
0Æ5 kW microwave radiated (filled circles), 2Æ0 kW microwave radiated
(open circles) or boiled (filled triangles), and then the protein content
was determined by the Bradford assay.
100
Nucleic acid leaked (µg ml–1)
80
60
40
20
0
0 20 40
Time (s)
Figure 3 The amount of nucleic acid released into the spore suspen-
sion of Bacillus licheniformis. The suspensions of isolated spores were
0Æ5 kW microwave radiated (filled circles), 2Æ0 kW microwave radiated
(open circles) or boiled (filled triangles) relative to the time sequence.
The nucleic acid content of the supernatants was measured at
260 nm using a UV spectrophotometer.
2Æ0-kW microwave-irradiated spores was 8Æ5-fold that
released from 0Æ5 kW microwave-irradiated spores, repre-
senting a significant difference between the two. Boiled
and untreated spores showed no significant difference in
the amount of nucleic acid released. The presence of
nucleic acid in the suspension indicates the release of core
components as a result of damage to the spore structure.
These results indicate that 2Æ0 kW microwave irradiation
inflicted greater damage to the spore structure than
0Æ5-kW microwave irradiation or boiling, which correlates
well with the disruption of spores observed by transmis-
sion electron microscopy (TEM) discussed below.
The loss of enzyme activity could be a good marker
indicating loss of cell function caused by cell damage and
cell death. CotA is exclusively localized in the outer coat
layer of the spore, where it might act as a classical laccase
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885
Spore killing mechanisms by microwave irradiation
0·8
Laccase activity (OD405)
0·6
0·4
60 80 0 20 40 60 80
Time (s)
Figure 4 The laccase activity of Bacillus licheniformis in PB suspen-
sion. The suspensions of isolated spores were 0Æ5 kW microwave
radiated (filled circles), 2Æ0 kW microwave radiated (open circles) or
boiled (filled triangles) relative to the time sequence. Laccase activity
was assayed using the ABTS substrate.
in the context of the multiprotein coat structure (Hullo
et al. 2001; Enguita et al. 2003). Laccase activity was not
detected in suspensions of vegetative B. licheniformis.
Wild-type spores had full enzymatic activity toward the
laccase substrate ABTS, whereas the enzyme activity of
the spores was completely lost after microwave irradiation
for 1 min. The spores retained full enzyme activity after
boiling for 1 min (Fig. 4).
60 80 Effects of microwave irradiation on the ultrastructure of
B. licheniformis
We prepared spore samples for TEM to determine
whether the protein and DNA leakage that we observed
resulted from damage to the spore ultrastructure. The
ultrastructure of B. licheniformis spores is shown in Fig. 5.
In general, starting from the outside and proceeding
inward, the spore layers include the exosporium, coats,
cortex, germ cell wall, inner membrane and central core
(Setlow 2006). Figure 5a shows the morphological charac-
teristics of normal purified spores. The spore was almost
ellipsoidal and did not possess an exosporium. The spore
coats showed the typical organization of coat layers, with
a lamellar inner coat and a thick, electron-dense striated
outer coat closely opposed to the inner coat. The inner
coat was composed of about eight layers. The outer coat
stained darker than the inner coat and had a more coar-
sely layered appearance. The normal spore contained a
thick, grey layer of cortex, which was the thickest layer of
the spore envelope. The compact core was surrounded by
an inner membrane.
Some changes in the spore ultrastructure were observed
after boiling (Fig. 5b). In contrast to the untreated spore,
boiled spores showed evident swelling of the outer coat
and degradation of the inner lamellar coat. The thick,
881
Spore killing mechanisms by microwave irradiation
(a) (b)
(c) (d-1)
(d-2) (e)
grey layer corresponding to the cortex of the untreated
spore disappeared, and swelling of the cortex was
observed. The margin of the spore core was somewhat
blurred because the inner membrane had disappeared.
Figure 5c shows the ultrastructural features of 0Æ5 kW
microwave-irradiated spores. One-third of one spore
maintained normal shape and size, but the other part of
the coat and cortex had ruptured.
As shown in Fig. 5d-1,d-2, spores exposed to 2Æ0 kW
microwave irradiation had more significant changes to the
ultrastructure than boiled spores. The microwave treatment
separated the inner coat from the outer coat and ruptured
the outer coat. Cortex hydrolysis and swelling were
observed, similar to that of boiled spores. Both sides of the
inner membrane were damaged and formed a dispersed
electron-light region at the damaged core area.
With regard to shape and size, autoclaved spores
(Fig. 5e) were similar to untreated spores. However, in
contrast to untreated spores, autoclaved spores exhibited
a degraded outer coat and distorted inner coat. The
structure of the cortex had no major alterations, but one
portion of the cortex was degraded. The spore core
appeared larger in the autoclaved spore than in an
untreated spore.
882 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885
S.-Y. Kim et al.
Figure 5 Electron micrographs of Bacillus
licheniformis spore. The spores were
suspended in PB, then were untreated
(a; 50 000 · 1Æ8), boiled (b; 80 000 · 1Æ8),
0Æ5 kW microwaved (c; 60 000 · 1Æ8), 2Æ0 kW
microwaved (d-1; 80 000 · 1Æ8, d-2;
40 000 · 1Æ8) and autoclaved
(e; 60 000 · 1Æ8).
Discussion
Our results and those of others indicate that microwave
ovens can be used to kill microbial pathogens, but not
for sterilization of spore-forming micro-organisms
within a short exposure time. Bacterial spores displayed
a higher resistance to microwave radiation than did
vegetative bacteria or bacterial phages (Park et al.
2006). Latimer and Matsen (1977) reported that micro-
wave irradiation of vegetative B. subtilis resulted in total
sterilization within 1 min, but the time necessary to
destroy B. subtilis spores was longer. Although the
microwave power level had no significant effect on
E. coli destruction (Yaghmaee and Durance 2005), less
time was required to reach the boiling point with high
output power than with low output power (Celandroni
et al. 2004). Therefore, we modified a microwave oven
to produce a power output of 2Æ0 kW, which allowed a
shorter sterilization cycle.
In our study, after microwave treatment of B. licheni-
formis spores for 1 min, the survival was exhibited a 0Æ17-
log reduction of 0Æ5 kW microwave irradiation and a
1Æ97-log reduction of 2Æ0 kW microwave irradiation.
However, exposure for 60 min or more was adequate to
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S.-Y. Kim et al.
insure sporicidal activity of the same spores. In contrast
to boiling and 0Æ5 kW microwave irradiation, a 2Æ0 kW
microwave treatment for 1 min did not completely kill
B. licheniformis spores, but killed most spores. The spore
inactivation rate with 2Æ0 kW was faster than that with
boiling or 0Æ5 kW.
Although controversy remains over the mechanisms of
microwave-induced death of nonspore-forming bacteria,
bactericidal mechanisms differ between microwave treat-
ment and boiling. Injury to Staphylococcus aureus cells
was greater after exposure to microwave irradiation at a
sublethal temperature than after exposure to conventional
heating (Dreyfuss and Chipley 1980). Microwave irradia-
tion induced morphological modifications of fungus cells;
however, no evidence was found in morphological alter-
ation of fungus after submersion in boiling water for the
same amount of time, even though cell death was
achieved (Rosaspina et al. 1994). Microwave irradiation
breaks DNA located deep in the phage core, whereas
moist heating of phage particles does not (Kakita et al.
1995). Changes in the structural integrity and permeabil-
ity of the cell membrane and cell wall might detrimentally
affect cell metabolism and lead to cell death.
Despite many studies on the effects of microwaves on
vegetative bacterial cells, few studies have examined the
sporicidal effects of microwave irradiation. The disintegra-
tion of the spore structure observed by electron micros-
copy showed that the ultrastructural alterations of
microwave-irradiated spores involve a mechanism differ-
ent from that of the effects of conventional heating such
as boiling or autoclaving (Belliveau et al. 1992; Vaid and
Bishop 1998; Celandroni et al. 2004). In this study, the
analysis of protein and DNA release and the loss of laccase
activity, together with the TEM evidence, demonstrated
that spore injury induced by 2Æ0 kW microwave irradia-
tion was significantly different from that attributable to
boiling. In contrast to the 0Æ5 kW microwave irradiation
and boiling, 2Æ0 kW microwave irradiation produced sig-
nificant leakage of protein and DNA from spores as a
result of altered spore structure, as observed previously in
vegetative bacterial cells after microwave irradiation (Woo
et al. 2000; Hong et al. 2004). Spores have comparable
amounts of protein in the spore coat and nucleoid struc-
tures. In Bacillus, the spore coat comprises c. 50% of all
the spore proteins (Driks 1999). Therefore, the presence of
proteins in the microwaved spore suspension indicates
that the spore coat was ruptured.
The loss of enzyme activity could be a good marker
indicating the loss of cell function caused by microwave-
induced cell damage and cell death. The outermost layer
of the spore coat has two morphologically distinct layers:
an electron-dense outer layer and an electron-translucent
inner layer. CotA is localized exclusively in the outer coat,
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 877–885
Spore killing mechanisms by microwave irradiation
where it may act as a classical laccase in the context of
the multiprotein coat structure (Hullo et al. 2001; Enguita
et al. 2003). Laccase activity clearly correlates with spore
count (Hirose et al. 2003). In this study, the laccase acti-
vity of spores was completely lost after microwave irradia-
tion for 1 min, whereas the spores retained full enzyme
activity after boiling for 1 min. The complete loss of lac-
case activity was the result of coat protein denaturation,
which contributed to coat rupture. Furthermore, our
TEM images showed that the inner and outer coat layers
of 2Æ0 kW microwave-irradiated spores had separated,
leading to coat rupture. It has been suggested that the
electromagnetic fields associated with microwave irradia-
tion may interact with the cell membrane, producing
reversible or irreversible changes in its permeability
depending on the field strength (Ponne and Bartels 1995).
The presence of nucleic acid in the spore suspension
indicates the release of core components upon damage to
the spore inner membrane. Significant fragmentation of
microwaved spores has been shown to be associated with
nucleic acid in spore suspensions (Vaid and Bishop 1998).
Moist heat treatment of spores does not cause DNA dam-
age but is often accompanied by inactivation of core
enzymes and rupture of the inner membrane permeability
barrier (Setlow 2006). The release of spore core compo-
nents suggests the disruption of the inner membrane, pos-
sibly correlated with spore death (Belliveau et al. 1992).
In this study, changes in cortex structure were
also observed in TEM images of thin sections. The fresh
B. licheniformis spores contained a thick, grey layer of
cortex, which was the thickest layer of the spore envelope.
TEM revealed the loss of almost the entire spore cortex
after 2Æ0 kW microwave treatment. Swelling of the cortex,
similar to that with boiling treatment, appeared to occur
in microwave-irradiated spores, but Celandroni et al.
(2004) did not observe cortex swelling in spores irradi-
ated with low-power (0Æ1 kW) microwaves. No modifica-
tion of the cortex was observed in our 0Æ5 kW
microwave-irradiated spores. Our results suggest that
2Æ0 kW microwave irradiation affects spore cortex hydro-
lysis and swelling.
There are conflicting reports on whether microbial
inactivation is due to heating or to the electromagnetic
energy itself (Jeng et al. 1987; Wu 1996; Yeo et al. 1999;
Sudrik et al. 2002). Microwaves are a form of electromag-
netic energy. Dipole interactions occur with polar mole-
cules. Many molecules (such as water, methanol, ethanol,
etc.) are electric dipoles, meaning that they have a
positive charge at one end and negative charge at the
other. The polar ends of a molecule tend to align them-
selves and oscillate in step with the oscillating electrical
field of the microwaves. Collisions and friction between
the moving molecules result in heating (Ponne and
883
Spore killing mechanisms by microwave irradiation
Bartels 1995). Vaid and Bishop (1998) have observed con-
ventional heating, even when prolonged, did not result in
the fragmentation of spores produced by microwaves
seen. They suggested that the fragmentation of spores
seen under the conditions used is presumably attributable
to an explosion of internal pressure generated within the
core. Sufficient water is obviously present in the core to
become excited and hence produce the destruction
observed.
We observed the spore coat rupture and the inner
membrane disruption with microwave irradiation. This
effect was not seen with boiling or autoclaving. This dif-
ference could be related to differences in types of spore
damage produced by microwave vs boiling treatment.
These results suggest that the electromagnetic energy may
cause the explosive rupture of the outer coat.
Acknowledgements
We thank Byeong-Soo Lim, a technician of electron
microscopy, for expert assistance with the electron
microscopy. This work was supported by the Korea
Science & Engineering Foundation through the Infection
Signaling Network Research Center (R13-2007-020-
01000-0) at Chungnam National University.
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