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PROPOSAL
MS4091 First Final Project
By:
Bill Hakim (13117106)
Supervisor:
Poetro Lebdo Sambegoro, M.Sc., Ph.D
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Approval Sheet
By
Bill Hakim
13117106
Main Adviser
Co-Advisor Co-Advisor
Dr.Ir. Ignatius Pulung Nurprasetio MSME Dr. Eng. Bentang Arief Budiman
NIP 195709211984031001 NIP 118110023
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Proposal of Bachelor Thesis
Bacterial Inactivation by Ultrasonication: Finite
Title Element Analysis of the Inactivation Mechanism Bill Hakim
of Escherichia coli
Major Mechanical Engineering 13117106
Faculty of Mechanical and Aerospace Engineering
Institut Teknologi Bandung
Abstract
In times of rapid population growth, food security is crucial to sustaining the population,
especially when the food reserve struggles to follow population growth. Food preservation by
sterilization is one of the answers to that challenge. Although food preservation technology
has been developed since the dawn of civilization, the common technique of sterilization is
the thermal treatment which has a lot of drawbacks to the food quality as well: the
deterioration of nutritional value and organoleptic properties.
Ultrasonication can be categorized as a prominent alternative for food preservation.
Ultrasonication has a significantly less effectiveness than conventional thermal treatment in
the terms of preserved food quality. Although ultrasonication efficacy has been proved and
gained traction in the food industries, the science on how the mechanism works is still
indeterminate. Thus, research toward the exact mechanism of bacterial inactivation is
proposed.
The proposed research is addressing simulations based on Abaqus/Explicit Dynamic
analysis. In this case, Escherichia coli is chosen as the model bacteria since its role in food
spoilage. The required data for the simulation—cavitation parameter and mechanical
properties of the bacteria cell wall—has been acquired from the literature review.
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Contents
Contents .................................................................................................................................... iv
Bibliography ............................................................................................................................ 19
List of Figures
Figure 2.1 Cavitation formation induced by ultrasonic wave[5] ............................................. 11
Figure 2.2 Graph of nondimensional pressure to nondimensional distance from the collapsing
buble [14] ................................................................................................................................. 11
Figure 2.3 Types of hydrodynamic cavitation [11] ................................................................. 12
Figure 2.4 Overview of cavitation as bacterial inactivation [11]............................................. 12
Figure 2.5 Stress/Strain Graph of B. subtilis at various relative humidity values [26]............ 14
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List of Tables
Table 4.1 Research Schedule ................................................................................................... 18
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Chapter 1
Introduction
1.1 Background
Since the transition of man from food forager into food producer occurs, a lot of food
preservation techniques had been developed from a simple process of drying food into the
modern process of thermal treatment [1] Food preservation has always been crucial for the
society to live sustainably. In ancient times where the food could only be produced in a few
months throughout the year and the production itself is not very resilient towards pests or
inclement weather, stockpiling food was tremendously significant. Presently in modern times,
where the population is growing rapidly and likely to exert “extreme pressure” on the existing
food systems [2], food preservation is crucial for food security as a whole [3]. A third of 1300
million tons of food produced are abruptly wasted or lost. With decent food preservation
techniques, this number could be reduced significantly by storing food effectively and
distributed without a great strain on expiration and contamination.
The ultrasonication process is done by objecting food into ultrasonic waves, which have
a frequency of higher than 20 kHz, therefore inducing the formation of bubbles. This formation
is called acoustic cavitation—principally different from hydrodynamic cavitation where the
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cavitation is caused by the acceleration of the fluid relative to the submerged bodies. The
bubbles collapse causes thermal, mechanical, and chemical effects [4]. High-frequency
ultrasonication is used to obtain the physicochemical properties of food such as acidity,
firmness, sugar content, and ripeness. Meanwhile, low-frequency ultrasonication is used to
change physicochemical properties of food by inducing pressure, shear, and temperature
difference in the medium and is capable to produce cavitations to inactivate bacteria in foods
[5]. Hence in the food preservation process, low-frequency ultrasonication is usually
implemented.
The term cavitation was coined in 1895 in a paper by Thornycroft and Barnaby [6].
They observed this phenomenon occurring on the backsides of propeller blades. It was Lord
Rayleigh that discovered that the collapsing cavitation bubbles could generate very high
pressure which was capable to induce irreparable damage on the blades [7]. The evidence of
the damage was then acquired initially by Harrison in 1952 and Güth in 1956.
The potential of ultrasonication was first discovered when sonar, which was
investigated for applications in anti-submarine warfare, was killing fishes nearby. Therefore,
the research toward its potential as a method for inactivating cells proliferated. The research
spanning from the 1960s to the 1990s discovered the fundamental of ultrasonication such as
the phenomenon that occurs during the process and its effect to the microbial cell [8]. The
current research tends to consider the practical implementation of ultrasonication, especially
for food preservation. The other renowned application of ultrasonication is for water treatment
[9] and viral inactivation [10].
Although the efficacy of the process had been proved, the variability of the results is
still not capable to be explained definitively. It is found that not only the bacterial type
influences the efficacy rate, the type of ultrasonication also significantly affects the result [11].
The result of ultrasonication is usually obtained empirically by counting the CFUs of the treated
medium or TEM images of the bacterial cell. However, the exact mechanism of how the
collapsing bubble affects the cell is not very clear. Whether the thermal, physical, chemical
effects or the combination of them would inactivate cells, it is still debatable on the various
published paper.
In this final project, the rupture of rod-shaped bacteria due to pressure wave caused by
bubble collapse will be simulated numerically. Escherichia coli is taken as the model bacteria
considering the bacteria’s role in food spoilage and its shape. This work expectedly will cast
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light on the bacterial inactivation mechanism and serves as an input for future optimization of
the ultrasonication technology.
• The mechanism of bacterial inactivation due to ultrasonication has not been simulated
yet.
• The variability of ultrasonication result seems to arise from parameters which determine
the cavitation characteristic. However, the way that each parameter influences the result
of ultrasonication is still debatable, precisely on rod-shaped bacteria such as E. coli.
1.3 Objectives
Regarding the research background, the objectives of this thesis are listed below:
• In the modelling process, bacterial cytoplasm and suspension liquid surrounding the
cell is defined as water.
• The cell wall is assumed to be homogenous based on the general mechanical
properties of the bacterial cell.
• The simulated load for the FEM is based on the previous work.
• The simulation is done on a single cell of Escherichia coli.
• The simulation only considers the pressure gradient manifestation as the load. Other
aspects including electrostatic forces, surface tension, chemical effect etc, is
neglected to simplify the simulation.
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1.5 Significance of the Study
From this thesis, hopefully, these outcomes would be fulfilled:
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Chapter 2
Literature study
2.1 Cavitation
Cavitation is a formation of small vapour bubbles (cavities) inside a homogeneous
liquid medium caused by a local pressure drop [12]. In general, two types of cavitation are
recognised: acoustic cavitation (AC) and hydrodynamic cavitation (HC). The difference
between the two is the mechanism that triggers the local pressure to drop.
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- Stable cavitation are bubbles that oscillate during many cycles of acoustic pressure
waves. They exist long enough to aggregate into large bubble clouds with some
equilibrium size.
- Transient cavitation are bubbles that exist for a short period, sometimes less than one
cycle, and collapse violently.
10 4
5
Nilai Tekanan Nondimensional dari Gelembung Kolaps (p * )
l
3.5
2.5
1.5
0.5
0
0 100 200 300 400 500 600 700 800 900 1000
* *
Jarak Nondimensional dari Gelembung Kolaps (r -R )
Figure 2.2 Graph of nondimensional pressure to nondimensional distance from the collapsing buble
[14]
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In HC, cavitation is caused by the acceleration of the fluid relative to the submerged
bodies. Flow conditions and geometry of the submerged body define the cavitation behaviour
and its characteristics. Generally, HC is divided into three categories: 1.) attached cavitation,
2.) cloud shedding cavitation, and 3.) supercavitation. The visualization of each categories can
be seen in figure III.3. The HC has no contribution in the ultrasonication process.
- Bacterial cell wall damage caused by the mechanical effects induced by pressure
gradient within or near the bacteria. The transient cavitation bubble would collapse
violently and produce a pressure gradient. This pressure gradient is manifested into
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three types of mechanical effects: shock waves, liquid microjets, and high shear forces
(turbulence and eddies) [16]. The collapse could release a shockwave which pressure
could reach up to 100 MPa [17]. If the bubble collapsed asymmetrically, microjets with
high velocities above 100 m/s is occurred [18]. Besides, the bubble collapse would also
causes microstreaming that damages the bacterial cell due to a highly localised shear
force [19].
- Shear forces in the bacterial cell that disrupting the inner organelle of the cell.
- Chemical attack due to the formation of free radical. Besides physical effect, the
cavitation bubbles collapse could form the so-called hot spots with extreme temperature
in the order of several 1600 K to 9000 K [20] which can cause the formation of radicals
from water molecule such as H+ and OH- [21].
The exact mechanism has not been determined yet. The variation between the three
classes is found in a lot of research. Ultrasonication may inactivate bacterial cells by
mechanical and/or chemical effects as mentioned above. The mechanism is also said to be
directly or indirectly dependent on processing variables such as ultrasound source and reactor
geometry, frequency, and acoustic energy density. Media properties including treatment
volume, temperature, viscosity, and gas concentration also affect the efficiency of inactivation
[13].
For E. coli, Hyoungill et. al has investigated the effect of pressure and thermal treatment
in ultrasonication, manosonication, thermosonication, and manothermosonication.
Manosonication is an ultrasonication process that introduce certain external pressure to the
medium while in thermosonication heat is added to raise the medium temperature. Meanwhile,
manothermosonication combines both of temperature and pressure. By varying the parameter
over a same span of time, they were able to plot the corresponding cell number after each
treatment after some period. Nonetheless, the parameter used asre still limited, as the paper
stated. Ultrasound parameter such as frequency, amplitude, energy density, and the properties
of the medium—which was not controlled in this research—should have played an important
role in the inactivation [22]. Debate between various paper on the result of the ultrasonication
seems to appear because of these parameters, where the experiments usually did not dwell too
much in it.
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Bacteria could be determined to be either gram-positive or negative. The determination
is based on its response to gram staining, a method that was established by Christian Gram in
1884. Both types differ on their cell wall’s structural composition. A typical gram-positive
bacteria’s cell wall is composed of layers of peptidoglycan, 20-80 nm thick, which lies outside
the plasma membrane, whereas gram-negative bacteria’s cell wall is composed of a thinner
peptidoglycan layer, 5-10 nm thick and an additional structure called the outer membrane, 7.5-
10 nm thick [23]. Physically speaking, gram-positive bacteria is stronger than gram-negative
bacteria. As a gram-negative bacteria, E. coli geometry is a cylinder 1.0-2.0 µm long, with
radius about 0.5 µm [24]. The thickness of its cell wall is at most 4 nm [25].
One of the most prominent research regarding the mechanical properties of the bacterial
cell wall is conducted by Thwaites and Mendelson in 1991 [26]. A thread of roughly 300 cells
of Bacillus subtilis was objected to tensile test. They were able to construct nominal
stress/strain curves for B. subtilis at four values of relative humidity shown in figure III.5
below.
Figure 2.5 Stress/Strain Graph of B. subtilis at various relative humidity values [26]
Even though B. subtilis is a gram-positive bacteria, the stress characteristic of the gram-
negative E. coli should be about the same. The thick cell wall of gram-positive bacteria is
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compensated by its higher turgor pressure. It is suspected that these factors sum up into the
same stress characteristic [26].
Inside the cell walls is a cytoplasm with organelles suspended in it. Organelles are
membrane-bound structures inside the cell that have several specific function. Considering the
composition of the cell contents, it could be categorized as water. This approach is considered
valid since water makes up about 80% of cytoplasm [27]. The cell surrounding is also assumed
to be fully water.
In this research, finite element analysis (FEA) is conducted by using software Abaqus.
Abaqus is embedded with the explicit analysis that will suffice the requirement for the modelled
phenomena. The explicit analysis takes account of the finite propagation speed of dynamic
effect throughout the material as opposed to implicit analysis. Hence, explicit analysis would
produce a more accurate result.
From the literature review above, substantial data for the research are obtained. The
parameter of ultrasonication and the bacterial cell wall is collected to construct the finite
element model of E. coli. The literature also confirms the previous claim that the research
regarding the exact mechanism of cell inactivation has not been modelled yet. It is expected
that this research would provide another insight into the inactivation mechanism.
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Chapter 3
Methodology
Based on the objectives mentioned before, the research method is built on parametric study
based on simulation using Abaqus. The simulation starts with modelling process where the
acquired secondary data from literature review will be the input for the bacteria model. The
model loads are varied according to the ultrasonication parameter. The simulation result is
compared visually with the TEM images to validates the simulation. Finally, the result is
assessed to determine each of ultrasonication parameter’s effect to the inactivation
performance.
• Literature review
The purpose of literature review is to gather data for the simulation and to set the
benchmark from the previous works. The required data is the mechanical properties of
the bacterial cell and the shockwave parameter of cavitation. Since this project is z
continuation of the previous one, the previous work will be set as the starting point and
reference.
• Modelling
The modelling is done by Abaqus software with the input gathered from the literature
review. Some assumptions due to the data limitation or to simplify the simulation
process. The process spans from building the geometrical model of the cell, assigning
the material, and setting its interaction to the other elements.
• Simulation
The finished Abaqus model is meshed into finite element sufficiently. After that, the
boundary condition is set. Then, the model is objected into a various load that is
considered as crucial in the ultrasonication process. Finally, the simulation job is
submitted to the software and would be ran iteratively to reach convergence.
• Validation
To ensure that the simulation would provide a representative result, a validation process
is necessary. The simulation result will be compared visually with the images from
transmission electron microscope. This comparison is done qualitatively to assess the
damage modes from both simulation and TEM images.
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• Simulation Analysis
Parametric study is conducted to assess the ultrasonication parameter influence to the
inactivation performance based on the simulation results. Parameter which would affect
the cavitation characteristic is varied to determine its relationship with the inactivation
performance.
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Chapter 4
Research Plan
The table below is the schedule of the research. It is based on the academic calendar of ITB
and presumptions of each process’s difficulty and duration.
Schedule Month
Activity 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Literature review
Modelling
Simulation
Validation
Simulation
analysis
Thesis writing
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