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Indonesian Journal of Science & Technology 6 (1) (2021) 65-80

Indonesian Journal of Science & Technology
Journal homepage: http://ejournal.upi.edu/index.php/ijost/

Bacterial Cell Inactivation Using a Single-Frequency
Batch-Type Ultrasound Device
Poetro Sambegoro1†*, Maya Fitriyanti2†, Bentang Arief Budiman1, Kamarisima2, Sekar Wangi Arraudah
Baliwangi1, Calvin Alverian1, Saeed Bagherzadeh3, Ganesan Narsimhan4, Pingkan Aditiawati2, Ignatius Pulung
Nurprasetio1*
1

Faculty of Mechanical and Aerospace Engineering, Institut Teknologi Bandung, Indonesia
2
School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia
3
School of Materials Engineering, Purdue University, Indiana, USA
4
Department of Agricultural and Biological Engineering, Purdue University, Indiana, USA
Correspondence: E-mail: poetrols@itb.ac.id, ipn@ftmd.itb.ac.id

ABSTRACT
Ultrasound technology employs cavitation to generate highpressure soundwaves to disrupt bacterial cells. This study
reveals the effectiveness of a single frequency ultrasound
device for bacterial cell inactivation. A low-cost ultrasound
device having a single frequency, i.e. 22 kHz for lab-scale
application, was developed first, and the prototype was
mechanically designed and analyzed using the finite-element
method to assure the targeted natural frequency could be
achieved. The prototype was then tested inactivating
bacterial cells, Escherichia coli (E. coli) and Bacillus subtilis (B.
subtilis), in a simple medium and a food system, and the
results were then compared to a commercial system. A
treatment time of up to 15 minutes was able to reduce E. coli
and B. subtilis cells by 3.3 log and 2.8 log, respectively, and
these results were similar to those of the commercial system.
The effectiveness of bacterial cell inactivation using the
developed single-frequency ultrasound device is then
discussed. The findings are useful for designing low-cost
ultrasound devices for application in the food industry.
© 2021 Tim Pengembang Jurnal UPI

ARTICLE INFO
Article History:
Submitted/Received 02 Oct 2020
First revised 08 Nov 2020
Accepted 13 Jan 2021
First available online 20 Jan 2021
Publication date 01 Apr 2021
____________________
Keyword:
Ultrasonication,
Cavitation,
Bubble,
Bacterial inactivation

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1. INTRODUCTION
Ultrasound
technology
has
been
developed for various applications, among
which are microbial cell inactivation and DNA
extraction (Chemat et al., 2011). Cavitation
occurs when vapor bubbles form in a fluid
due to a drop-in pressure below the
saturated pressure (Triawan et al., 2019).
Using ultrasonic technology, this pressure
drop is provided by a fluctuating transducer.
After the bubbles are formed, they undergo
an expansion and compression phases and
collapse. The collapse of the cavitation
bubbles can exert a high pressure due to the
shock wave and microjet effects. The
bursting cavitation bubbles have a highly
destructive effect that can be used to
damage bacterial cell walls. Water molecules
in the vicinity of the bubbles also exert a
shear stress effect and cause deformation of
the cell membrane. At the same time, the
local temperature also rises sharply. These
complex phenomena contribute to the
overall bacterial inactivation.
Several independent studies on bacterial
inactivation using ultrasound have been
performed. Ultrasonic waves generate the
bubbles and drive them to explode, resulting
in high pressure and temperature, and active
species in some cases (Wu & Nyborg, 2008).
Shear force due to an ultrasonic pressure
wave is commonly known to be the primary
cell-inactivation mechanism leading to cell
disruption and death (Shengpu Gao et al.,
2014a; Liao et al., 2018; Piyasena et al.,
2003). At higher frequency ranges, this
physical effect may be accompanied by a
sonochemical reaction in the surrounding
medium triggered by the increasing
temperature and pressure from the
collapsing bubbles (Ashokkumar, 2011).
Heat treatment is the most conventional
method applied to bacterial cell inactivation
in the food industry (Abdurrahman et al.,
2019; Chemat et al., 2011; Piyasena et al.,
2003; Raso & Barbosa-Cánovas, 2003).
However, the intensity of the heat treatment
may have several negative impacts on food

quality and nutrition (Chemat et al., 2011).
Another common method to inactivate
bacteria is by adding chemical preservatives.
This method may be effective but some
adverse side effects might not be tolerable in
the long term. Alternative approaches such
as application of natural antimicrobial
molecules (Brogden, 2005; Lyu et al., 2019;
Xiang et al., 2016) and non-thermal
technologies (pulsed electric fields, UV light,
high hydrostatic pressure, microwave,
ultrasound) have been explored to inactivate
cells (Butz & Tauscher, 2002; Leonelli &
Mason, 2010; Raso & Barbosa-Cánovas,
2003).
Unlike heat or chemical treatment,
mechanical treatments by using membrane
filter (Bilad, 2017; Gao et al., 2018; Sharpe et
al., 1979) and ultrasound can minimize
negative impacts in food quality and prevent
harmful health effects (Chemat et al., 2011).
While the membrane filter requires high
maintenance and does not inactivate the
bacteria, the ultrasound treatment has
proven to inactivate the bacteria effectively.
Ultrasound treatment especially for food
preservation can also be assisted with other
physical methods such as high pressure
(manosonication), heat (thermosonication)
and
combination
of
both
(manothermosonication) to enhance its
effectiveness (Piyasena et al., 2003; Sango et
al., 2014; Zupanc et al., 2019). Although,
these ultrasounds assisted technology is not
within our scope of experiment, but several
studies have mentioned their promising
results for milk and juices (Bermúdez-Aguirre
et al., 2009; Walkling-Ribeiro et al., 2009).
The physical parameters of the ultrasound
device are important to achieve maximum
inactivation effect. Working frequency and
intensity are important parameters that can
influence the cavitation effect in the
propagating media (Brotchie et al., 2009).
The readily available commercial
ultrasound device for cell disruption is
normally operated at a single frequency of 20
kHz. However, several studies showed that
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frequencies higher than 20 kHz cause more
effective biocidal effects (Shengpu Gao et al.,
2014a; Shengpu Gao et al., 2014b). This
motivated us to develop a low-cost
ultrasound device prototype with a
configuration that allows for different
frequency ranges. We designed a batch-type
ultrasound device installed with a
piezoelectric of 22 kHz.
The batch was designed to have a natural
frequency of 22 kHz to ensure that the
resonance phenomenon occurs. The
prototype was then tested on Escherichia coli
and Bacillus subtilis in a simple medium (e.g.,
phosphate buffer) and a more complex
medium (e.g., milk). The results were also
compared to those from another experiment
using a commercial-probe ultrasound device.
Parameters affecting the bacterial cell
inactivation
were
comprehensively
discussed.

2. METHODS
2.1. Ultrasound prototype design and
manufacture
The ultrasound prototype presented in
this study was of a tubular configuration that
had been developed previously (Borthwick et
al., 2005). Prior to manufacturing, vibration
analysis had to be conducted to assure the
prototype could vibrate with the maximum
amplitude at the designated frequency,
which directly corellated with the prototype
efficiency.
The vibration analyses, consisting of
modal analysis and harmonic analysis, were
performed using the finite element method

(FEM). Modal analysis was conducted to get
the characteristics of the system, including its
natural frequency and corresponding
vibration modes (Eslaminejad et al., 2019).
These parameters are the foundation for
harmonic response analysis, which results in
a response at every determined frequency.
The peak response corresponds to the
natural frequency of the system.
The system was a cylinder with one closed
end designed using the ANSYS Design
Modeler. The cylinder tank had an outer
diameter 45 mm, inner diameter (Di) of 25
mm, height of 22 mm, and hole depth of 18
mm Figure 1. The tank, which was made of
steel, was then fitted to a 22 kHz
piezoelectric transducer (Steiner & Martins
Inc, Doral, FL) and had the mechanical
properties presented in Table 1. The steel
material was selected because it can convert
mechanical vibration generated by the
transducer to be pressure waves in liquid
inside the tank with low damping
(Nurprasetio et al., 2018).
A pressure load to substitute the
transducer contact was applied to the outer
perimeter of the cylinder surface as the
loading condition, which can be seen in
Figure 2. The magnitude of the pressure was
obtained by dividing the maximum force of
the piezoelectric with its surface area; in this
case, 0.25 MPa was input into the system.
The settings used for harmonic response
analysis are shown in Table 2. The overall
system consisted of a signal generator (Rigol
DG 812), a power amplifier (PCA MOS 4175),
an oscilloscope (GW Instek GDS 1054B), and
a transformer (ING 10A 110 240). Figure 3 is
a schematic of the circuit of the experimental
apparatus.

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Figure 1. CAD model of batch type ultrasound prototype

Table 1. Mechanical properties of steel used for ultrasound prototype material
Mechanical Properties
Density
Young’s Modulus
Poisson Ratio
Bulk Modulus
Shear Modulus

Value
7800 kg/m3
200 GPa
0.3
166.7 GPa
76.9 GPa

Figure2. Boundary condition for harmonic response simulation

Table 2. Input data of harmonic response analysis
Parameter
Frequency Spacing
Minimum testing frequency
Maximum testing frequency
Solution Intervals
Solution Method

Value(s)
Linear
0 Hz
40000 Hz
40 (0-40 kHz)
200 (36-38 kHz)
Mode Superposition

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Small Resistor
Piezo

Transformer

Figure 3. Electrical resonance circuit of the experimental apparatus
2.2 Bacterial cell preparation
E. coli and B. subtilis cultures were
provided by the Microbiology Laboratory at
the School of Life Sciences and Technology at
ITB. Each culture was grown overnight in
Luria Broth media (HiMedia, Mumbai, India)
at 37 °C, and it reached 6.5-7 log colonyforming unit per mililiter (CFU/ml). The
culture was then harvested and centrifuged
down at 10,000 g for 5 minutes. The pellet
was washed twice using a phosphate buffer
saline solution at a pH of 7.4. The final
suspension with the same buffer was used
for the ultrasound treatment.
2.3 Food sample preparation
Commercial pasteurized cow milk (full
cream) was used as a sample. The milk, with
a pH of 6.5, was obtained from a local
supermarket. Prior to ultrasound treatment,
two batches of pasteurized milk were each
inoculated with E. coli and B. subtilis and
incubated overnight at 37 °C until they
reached 6.5-7 log CFU/ml. The inoculated
milk was stored at 4°C before the
experiment.
2.4. Ultrasound experiment
Ultrasound treatment was carried out
using the 20 kHz prototype and a 20 kHz

probe cell disruptor (Omni Ruptor 4000, GA,
USA) for comparison. Five ml of bacterial
suspension in pH 7.4 PBS and inoculated milk
were exposed to ultrasound for 1, 5, 10, and
15 minutes. The probe cell disruptor was set
at 20% and 40% power output (equal to 80W
and 160W) with constant sonication. To
determine number of surviving cells after
treatment, 0.1 ml of each treated sample was
spread in Luria Agar (HiMedia, Mumbai,
India) and grown at 37 °C for 12 – 16 hours
until the colony was viable.
2.5. Microscopic analysis
To study cell agglomeration of B. subtilis
before and after ultrasound treatment, cell
suspension was analyzed under an inverted
phase contrast microscope (Nikon Eclipse TE
300, NY, USA). The culture suspension was
dropped onto a microscope slide (10-20 l)
using a glass slide cover. Visualization was
performed at 10x, 20x, and 40x
magnification.
2.6. Genomic analysis
Extraction of genomic DNA of E. coli and B.
subtilis after ultrasound treatment was
performed according to Rapley. The
extracted DNA was then amplified using a
mini8 thermal cycler (miniPCR, MA, USA)
with a temperature cycling consisting of a
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denaturation stage at 94 oC for 5 min,
followed by 30 cycles of 94 oC for 20s, 60 oC
for 30s and 72 oC for 30s, and a final stage at
72 oC for 10 min (González-González et al.,
2019). After amplification, the genomic DNA
was loaded onto a 1.5 % agarose gel with 100
V for 25 min. The DNA bands were visualized
with long-wave UV light.
3. RESULTS AND DISCUSSION
3.1. Modal analysis of ultrasound prototype
Natural frequencies of modal analysis
obtained from finite-element analysis are
presented in Table 3. The first six modes
were rigid body movement, which means the
tank did not undergo any significant internal
deformation. The rest of the modes were the
bending modes. Two pairs of modes, 7th and
8th, as well as 9th and 10th, each had the same
natural frequencies, however they had
different phases. This was due to the
symmetric geometry of the tank.
The 7th and 11th modes show bending
oscillation in axial and radial movement as
presented in Figure 4. These modes follow
the loading condition from the piezoelectric
(cyclic radial force on the outer surface of the
tank).
Modal analysis Figure 5 of a tank with a 25
mm inner diameter (Di) shows the maximum
radial displacement (7.8 x 10-3 mm) that
occurs at 37 kHz. Ideally, the prototype
should be operated at this frequency to
ensure high-power conversion efficiency.
However, one of the constraints in the design
was the availability of the commercial
piezoelectric transducer.
In this work, we limited our work by
designing the prototype using a piezoelectric
transducer with a working frequency of 22
kHz. Due to this constraint, the radial mode
needed to be lowered from 37 kHz to 22 kHz.
This could be achieved either by lowering the
stiffness or by adding mass. The first option
proved to be simpler. The stiffness of the
tank was lowered by decreasing the wall
thickness between the inner and outer

surfaces. This parametric study was
performed by increasing the inner diameter
(Di) by 1 mm increments until the natural
frequency was found to be 22 kHz.
The natural frequency of 22 kHz was
obtained when the inner diameter of the
tank was 35 mm (23kHz) to 37 mm (21 kHz).
For higher frequency accuracy, the resolution
of the harmonic response was reduced to
100 Hz, and the harmonic response was
conducted between 20 kHz to 25 kHz to
shorten processing time. The increment of
inner diameter (Di) was reduced to 0.1 mm to
improve accuracy. The parametric study is
presented in Figure 6. The results show that
an inner diameter of 35.7 mm gives a natural
frequency of 22 kHz; consequently, 35.7 mm
was chosen as the inner diameter of the tank.
A linear relationship was observed between
the natural frequency and inner diameter for
the range of 25 to 38 mm. This indicates that
a typical batch can be designed and
manufactured
for
different
natural
frequencies by simply changing the inner
diameter.
Harmonic response analysis was repeated
to analyze the tank displacement response
between 0 and 40 kHz using the new inner
diameter. The resolution was 1 kHz, but near
the resonance frequency area, the resolution
was lowered to 10 Hz. Moreover, the
frequency response of tank stress (normal
stress in the radial direction) was also
conducted to evaluate the ability of the tank
to withstand vibration at the resonance
frequency.
The displacement and stress at 22 kHz
were 1.7 x 10-3 mm and 5.7 MPa,
respectively. The displacement was 100
times higher than that of the first design.
Maximum displacement and stress occurred
at 22.04 kHz with 5.5 x 10-3 mm displacement
and 18.5 MPa. Maximum stress was found to
be only 18.5 MPa, which was lower than the
fatigue strength; thus, the tank could have an
infinite lifetime. The FEM analysis ensures
that the prototype can be built and used for
the experiment.
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Table 3. Natural frequencies and their modes
Mode
1
2
3
4
5
6
7
8
9
10
11
12

Frequency (Hz)
0
1.47e-2
2.11e-2
0.14024
0.14754
0.15828
19999
19999
34175
34175
37634
40436

Type of Mode
Rigid body movement
Rigid body movement
Rigid body movement
Rigid body movement
Rigid body movement
Rigid body movement
Bending
Bending
Bending
Bending
Bending
Bending

Figure 4. Bending mode shapes of the tank under (a) 20000 Hz (7th mode) and (b) 37540 Hz
(11th mode)

Figure 5. Maximum displacement of the tank for all tested frequencies
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Figure 6. (a) The natural frequency alteration due to inner diameter changes. (b) A natural
frequency of 22 kHz when the inner diameter was changed to 35.7 mm. (c) The maximum
displacement of the tank for a frequency of 22 kHz.
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3.2. Manufacturing and prototyping
The cylinder was machined using a
standard milling and drilling machine with
100 m accuracy. This accuracy is required to
ensure a natural frequency of the cylinder of
around 22 kHz. The piezoelectric transducer
was force fitted on the outer surface of the
cylinder. Since the piezoelectric has an
excited frequency of 22 kHz, the cylinder
resonates and causes abundant bubble
generation. The system was then connected
to a function generator and an amplifier to
enhance the signal.
3.3 Microbiological and genomic DNA
analysis
Figure 7 shows the reduction of viable cell
numbers (cell densities) after being treated
with a probe cell disruptor. The treatment
was carried out at different ultrasonication
time periods and power levels. In general,
the results indicate a decline in cell numbers,
with E. coli being more sensitive to
ultrasonication compared to B. subtilis. After
a 15-minute sonication at 160 W Figure 7a,
the cell density was reduced by 3.6 log for E.
coli while B. subtilis was reduced by 3.4 log.
At 80 W for 15 minutes, the cell density was
reduced by 3.4 log for E. coli and 3.0 log for
B. subtilis Figure 7b. The prototype, with a
lower power level of 8 W and a 15-minute
exposure time, reduced E. coli and B. subtilis
cell densities by 3.3 log and 2.8 log,
respectively Figure 8. This result was
comparable to treatment with a probe cell
disruptor at 80 W. The prototype succesfully
replicated the tendency of experimental
results from commercial probes.
For treatment of bacterial cells in a more
complex matrix such as milk, both
commercial probes and our prototype show
less inactivation sensitivity. For the
commercial probe, cell density was reduced
by up to 3.0, whereas for our prototype, the
cell density reduction was up to 2.5 log. The
different inactivation sensitivities with

respect to the medium compositions and
viscosities were ultimately influenced by
acoustic transfer and distribution of power
within the reaction solution (BermúdezAguirre et al., 2009; Fitriyanti & Narsimhan,
2018; Joyce et al., 2011). In the case of milk
composition, the lower efficiency at the
current treatment intensity might have been
due to milk proteins and cell agglomeration,
which provided protection against biocidal
attack.
It can be seen in Figure 7a and 7b that
both power level and exposure time increase
cell inactivation. This has also been widely
observed in other studies (Bermúdez-Aguirre
et al., 2009; Shengpu Gao et al., 2014; Joyce
et al., 2011; Joyce et al., 2003; Sango et al.,
2014; Tiwari et al., 2009). A study conducted
by (Joyce et al., 2011; Fülscher & Roos, 1994)
observed that ultrasound treatment with an
intensity of 0.24 W/cm3 (20 kHz probe
sonicator) is more effective in reducing B.
subtilis cells than treatment with an intensity
of 0.18 W/cm3 (38 kHz batch sonicator).
Frequency and power level are two
important factors in determining the effect
of ultrasound because they influence the size
and number of cavitation bubbles (Joyce et
al., 2011). It has been found that at higher
frequencies (>500 kHz) and lower power
levels, cavitation bubbles are smaller and
collapse with less energy due to a shorter
acoustic cycle, which means a shorter time
for bubble formation. As a consequence of
this phenomenon, a bacterial suspension
treated with a higher frequency and a lower
power level experiences a significantly lower
mechanical effect (Joyce et al., 2011).
The difference in sensitivity between the
two tested bacterial cells, E. coli and B.
subtilis, may be due to differences in their
cell wall characteristics. As mentioned in
previous studies, gram-negative bacteria
with thinner cell walls such as E. coli, E.
aerogenes, and K. pneumoniae are more
sensitive to ultrasonication than grampositive bacteria with thicker cell walls such
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as B. subtilis and A. pullulans (Fitriyanti &
Narsimhan, 2018; Shengpu Gao et al., 2014;
Joyce et al., 2011). It has also been observed
that cocci, or spherical bacteria, are more
resistant than bacilli, or rod shaped bacteria
(Joyce et al., 2011). This corresponds to our
experimental results, where E. coli cell
reduction was higher than that of B. subtilis.
While Figure 7 shows the predicted trend in
terms of treatment time, the inactivation
rate seems to vary with differing time
periods. This may be due to the
agglomeration of the bacterial cells. Fülscher

reported that ultrasound treatment (20 and
38 kHz) for 1 to 10 minutes did not have a
dramatic effect on the cell viability, except
for a small drop after 15 minutes of
treatment. This insignificant result suggested
a declumping of bacterial agglomerates with
little deactivation. We also observed similar
agglomeration as shown in Figure 9.
Agglomeration of cells or biofilm formation
were indicated in untreated B. subtilis
suspension. After 15 minutes of treatment, a
declumping of bacterial cells occurred,
resulting in distinct individual cells.

Figure 7. Viable cell reduction of E. coli and B. subtilis after treatment with probe ultrasound
(20 kHz) using different input power levels: (a) 80 watt and (b) 160 watt. N = cell number
after treatment, N0 = initial cell number. Experiment kept at room temperature with
constant sonication

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Outside
the
conventional
power
ultrasound frequency range (100 kHz <
frequency < 2 MHz), acoustic cavitation may
induce mechanical effects and sonochemical
reactions (production of free radicals) that
can damage DNA, enzymes, liposomes, and
membranes (Butz & Tauscher, 2002;
Shengpu Gao, Hemar, et al., 2014; Wu &
Nyborg, 2008). As observed by Gao et al.
(Shengpu Gao et al., 2014) ultrasound at 850
kHz was highly efficient in inactivating
bacteria such as (B. subtilis, Enterobacter
aerogenes, Staphylococcus epidermidis) and
yeast (Aureobasidium pullulans) with
inactivation rates of up to 99%. The
inactivation is due to mechanical effects and
generation of hydroxy radicals that cause
damage to DNA and proteins. Based on these

studies, we performed a genomic DNA
analysis to examine the effect of prototype
power intensity on bacterial cell DNA. Figure
9 presents genomic DNA bands of E. coli (B
and C) and B. subtilis (D and E) after
treatment with our prototype.
The
fluorescent density of DNA bands decreased
with longer ultrasound treatment time.
Based on these results, the cells
disintegrated, causing DNA molecules to be
detected in the surrounding medium using
gel electrophoresis. The results were
consistent with another report which also
observed a decrease in DNA bands for cells
treated with longer sonication (Liao et al.,
2018).

Figure 8. Viable cell reduction of E. coli and B. subtilis after treatment with prototype
ultrasound (22 kHz). N = cell number after treatment, N0 = initial cell number. Experiment
kept at room temperature with constant sonication

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Figure 8. Phase contrast microscopy of B. subtilis. (a) Untreated culture. Red arrow shows
agglomeration/biofilm formation. (b) After treatment with the ultrasound prototype for 15
minutes. Red box shows declumping of cells. (c) Closer look at cells from panel B shows
distinct cells.

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Figure 9. Agarose gel electrophoresis of genomic DNA from bacterial cells treated with
prototype ultrasound for 5 and 10 minutes. A = marker, B = B. subtilis (5 min), C = B. subtilis
(10 min), D = E. coli (5 min), E = E. coli (10 min)
4. CONCLUSION
The readily available ultrasound probe
used in the lab for cell disruption works
mainly at 20 kHz. This limitation creates an
opportunity for exploring a low-cost
prototype. Due to the constraints imposed by
the availability of piezoelectric transducers,
the prototype geometry was optimized by
changing the tank’s inner diameter. The peak
resonance with a frequency of 22 kHz was
obtained for a diameter of 35.7 mm. The
relationship between the natural frequency
and the inner diameter of the tank was
revealed. This current prototype design will
later allow further modifications such as the
use of transducers of different frequencies
and a continuous flow-through system.
The current ultrasound prototype was
able to inactivate bacterial cells, E. coli and B.
subtilis in a simple medium (PBS, pH 7.4) and
in a food system (milk, pH 6.5). The results
were comparable to treatment with a
commercial probe cell disruptor. From the

experiment, it was confirmed that B. subtilis
has more resistance to cavitation bubbles
generated by an ultrasonicator than E. coli.
The experiment also confirmed that the cell
density reduction in milk is lower. Further
biochemical analysis and ultrasound power
intensity variations need to be performed to
understand this phenomenon better.
5. ACKNOWLEDGEMENT
This research was supported by the
Institute of Research and Community
Service, Institut Teknologi Bandung, under
the 2020 Multidisciplinary Research
Program.
6. AUTHORS’ NOTE
The authors declare that there is no
conflict of interest regarding the publication
of this article. Authors confirmed that the
paper was free of plagiarism.

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