electrophoretic studies of surface charge on unicellular bacteria
TRANSCRIPT
ELECTROPHORETIC STUDIES OF SURFACE CHARGE ON UNICELLULAR BACTERIA
LEE POH FOONG
FACULTY OF SCIENCE UNIVERSITY OF MALAYA
KUALA LUMPUR APRIL 2009
ELECTROPHORETIC STUDIES OF SURFACE CHARGE ON
UNICELLULAR BACTERIA
LEE POH FOONG
DISSERTATION PRESENTED FOR THE
DOCTORATE OF PHILOSOPHY
FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR
APRIL 2009
ACKNOWLEDGEMENTS
There are several people that have positive influenced me during my postgraduate
years and I would like to acknowledge them here. First of all, I would like to thanks to
my supervisor, Prof. Wan Ahmad Tajuddin for giving me a challenging and interesting
research project. This project has provided me a chance to venture into combination
fields which comprises of biology, chemistry and physics. Beside, I am thankful to his
motivation, encouragement and support these few years. Furthermore, I would like to
thank him for taking me as his student and has given me with sufficient freedom to
accomplish this project.
To my co-supervisor, Prof. Madya. Dr.Misni Misran from Chemistry Department,
University Malaya., I would like to convey my appreciation on his concern on students’
progress. This is really helpful and important on keeping a project moving forward. I am
also grateful for his willingness to spend plenty of his precious time to discuss on my
work. Thanks to his patient and constant encouragement throughout my study.
Special thanks to Prof. Thong Kwai Lin from Microbiology Department, University
Malaya, who has allowed me to use the facilities in Microbiology Lab and arranged a
very helpful final year student to help me out on my project. I have a great time with her
student name, Fong Bao Wen. She has shared lots of knowledge on handling the
bacteria with me. Furthermore, thanks to her for sharing a friendship and her valuable
help.
I would like to acknowledge the various companies that have provided me the
fruitful training on operating instruments in Colloid Lab, University Malaya. I
appreciate my friends from colloid lab who share wonderful time together throughout
these few years.
I also would like to thank to Mr.Paul Davidson for his efforts to proofread this
thesis. To all my friends, thanks for always be my side to support and encourage me
throughout the years. This is a very great moral support to go through tough time.
I am extremely grateful to my mother who has raised me up and provided me the
chance to study. She has also given me the confident and dedication to complete the
mission in life.
To my dearest husband, Bryan Kok, whose encouragement, love, cares, support,
motivation and accompany that has never dried out to me. Thanks for his love and
inspiration that have added another dimension to my life. Without him, the
accomplishment of this project is impossible. Lastly, not forget to dedicate this
acknowledgment to my dear girl, Minnie Kok. Thanks to her for being my girl.
Thank you.
ABSTRACT
Electrophoretic studies of surface charge on unicellular cell were carried out.
The unicellular bacteria selected for this research consisted of two different strains,
which are gram-positive S.aureus and gram-negative E.coli. Colloidal particles,
(Titanium dioxide) TiO2, and liposome, were used as non-living counterparts for
comparison. The studies have focused on the effects of different physical and chemical
conditions, including variations of temperatures, applied field, and time intervals
between measurements. We did calculations of the Donnan potential and also
investigated the polarizability of the bacteria. Methodology used in this research was
laser Doppler velocimetry electrophoresis. Surface charge of bacteria cells is measured
with electrophoresis due to development of net charge at the particle surface affects the
distribution of ions in the surrounding interfacial region. This process increases
concentration of counterions (ions of opposite charge to that of the particle) close to the
surface. Zeta potential is considered to be the electric potential of this inner area
including this conceptual "sliding surface". The electrophoretic mobility ( μ ) is the
migration rate of the charged particles to the electrode when electric field is applied. In
various pH, dead cells of both E.coli and S.aureus exhibited lower electrophoretic
mobility than live cells. This was mainly due to dead cell had lost the metabolic activity
to give charge balance to surface charges. Besides, it was found that increase in the
ionic strengths of NH4Cl in the buffer reduced the electrophoretic mobility of samples.
In contrary, increased ionic strengths of NaCl obtained the opposite result for the
bacteria. This might be due to the increase of influx Na+ into cells, which increases the
anionic lipid to balance the charges in the cells. Results indicated that both approaches
fitted well at greater ionic strength of NaCl with the Donnan potential approximation.
The electrophoretic mobility of live E.coli displayed a mild increase at higher
temperatures. On the other hand, results indicated only a slight increase in negative
electrophoretic mobility of live S.aureus as the temperature increased. Increase in
applied field increased the electrophoretic mobility of bacteria and colloidal particles
due to the stronger attraction field. Different time intervals between measurements with
unchanged conditions showed that higher zeta potential was measured for time intervals
more than 10s between measurements. This indicated that the particles still swirled
around when the next measurements started. In addition, live bacteria recorded greater
polarizability compare to colloidal particles. This may be attributed to the
semipermeable cell membrane of live cells which hasten the ions exchange between the
cell interior and the external environment. A greater polarization of E.coli was obtained,
which possessed thinner cell wall peptidoglycan layer compared to S.aureus. The
conductivity and permittivity of cells increased as the frequency increased. However,
the permittivity of cells were shown constant at higher frequency (>200Hz).
Keywords : Electrophoretic mobility; peptidoglycan layer; surface charge
ABSTRAK
Kajian ini adalah berkaitan dengan permukaan cas pada membran unisel. Mobiliti
elektroforetik daripada bakteria jenis gram-positif dan negatif adalah diukur dengan
Laser Doppler Velocimetri Elektroforesis Sistem. Kesan kepada permukaan cas
daripada perubahan jenis kimia elektrolit adalah dikaji. Kesan kimia yang dikaji
termasuk pH, pelbagai kemolaran ion daripada NaCl dan NH4Cl, centrifugasi dan
rendaman dalam air suling. Selain itu, kajian ini juga termasuk kesan permukaan cas
terhadap perubahan suhu, kesan medan elektrik, pelbagai masa selang ukuran and beza
keupayaan Donnan dikira dan turut dibincang dalam kajian ini. Penyelidikan ini adalah
penting untuk membezakan mobiliti elektroforetik untuk bacteria jenis gram-negatif and
positif dan juga termasuk bakteria yang hidup dan mati. Tambahan pula, mobiliti
elektrophoretik zarah-zarah koloid diukur untuk berbanding dengan sel biologi.
Keputusan daripada kajian ini mendapati bahawa centrifugasi akan menambahkan
elektroforetik mobiliti. Di samping itu, S.aureus dan E.coli yang mati turut memperoleh
mobiliti elektroforetik yang lebih rendah dalam pelbagai pH. Semakin tinggi kemolaran
NH4Cl, semakin turun elektroforetik mobiliti. Ini bercanggahan dengan keputusan
kenaikan kemolaran NaCl. Elektroforetik mobiliti kedua-dua bakteria menepati
pengiraan teori Donnan. Elektroforetik mobiliti bahan kajian ini turut meningkat dalam
kenaikan suhu and voltan. Sela masa antara pengukuran elektroforetik mobiliti bahan
pada 10 saat memberi keupayaan zeta yang lebih tinggi. Di samping itu, bakteria hidup
memberi keputusan polarasi yang lebih tinggi daripada bahan koloid. Ini adalah kerana
bakteria mempunyai sel membran yang mengizinkan laluan zarah-zarah kecil.
CONTENTS
Chapter Page
Acknowledgements ii
Abstract iv
Abstrak vi
Contents vii
List of Figures xii
List of Tables xvi
List of Abbreviations xvii
Chapter 1 : Introduction
1.0 Electrophoretic Studies of Surface Charge on Unicellular
Bacteria
1
1.1 Literature review 3
1.2 Scope of the Study 6
1.3 Outline of the Thesis 8
Chapter 2 : Characterization Of Surface Charge Bacteria
2.0 Introduction to Gram-Positive and Negative Bacteria 9
2.1 Escherichia coli and Staphylococcus aureus for Surface
Charge Study
12
2.2 Colloidal Particles as a model of Biological Cells 13
2.3 Negative Surface Charge of Bacteria and Colloidal Particles 14
2.4 Summary of Surface Charge
18
Chapter 3 : An Approach To Surface Charge And Electrical Double Layer
3.0 Introduction to Surface Charge and Electrical Double Layer 20
3.1 Origin of Charges at a Surface 20
3.2 Introduction to Electric Double Layer 21
3.3 Introduction to Zeta Potential 25
3.3.1 Electrokinetic Phenomenon: Electrophoresis 25
Chapter 4 : Methodology And Materials
4.0 Preparation of the Materials (Colloid Particles and Bacteria) 32
4.1 Solvent and Buffers 32
4.1.1 Preparation of TiO2 32
4.1.2 Preparation of Fatty Acid Liposome 33
4.1.3 Preparation of Live E.coli and S.aureus 33
4.1.4 Preparation of Dead E.coli and Dead S.aureus 34
4.2 Introduction to Laser Doppler Velocimetry Electrophoresis System
34
4.2.1 Overview on Electrophoretic Light Scattering 36
4.2.2 Advantages of LDV to overcome Electroosmosis Effect
39
4.2.3 Introduction on M3-PALS Technique 40
4.2.4 Operation of Zetasizer for Mobility Measurement 41
Chapter 5 : Effects Of Different Chemica l Environments On The Electrophoretic Mobility
5.0 The magnitude of the electrophoretic mobility of bacteria and colloidal particles in different chemical environment and salt effects
44
5.1 Size distribution of E.coli and S.aureus 45
5.1.1 Size distribution of TiO2 and Liposome 46
5.2 Effects of Number of Washes on Surface Charge of live E.coli and S.aureus
47
5.3 The Magnitude of the Electrophoretic Mobility of Bacteria and TiO2 in Distilled Water
48
5.4 The Electrophoretic Mobility of Biological Cells and Colloidal Particles in Different pH
50
5.4.1 Comparison of the Magnitude of the Electrophoretic Mobility of Live and Dead E.coli (TiO2 as reference)
51
5.4.2 Comparison of the magnitude of the electrophoretic mobility of live and dead S.aureus (TiO2 as reference)
53
5.4.3 Comparison of the magnitude of the electrophoretic mobility of live E.coli and S.aureus at different pH
56
5.4.4 Conclusion on the effects of pH on surface charge of live and dead bacteria (TiO2 as reference sample)
56
5.5 Effects of different salt on the magnitude of the electrophoretic mobility of live and dead E.coli and S.aureus (TiO2 as reference sample).
58
5.5.1 Effects of ammonium chloride (NH4Cl) on the magnitude of the electrophoretic mobility of TiO2 and bacteria
60
5.5.2 Effects of NH4Cl on the magnitude of the electrophoretic mobility live and dead E.coli (TiO2 as reference sample)
62
5.5.3 Effects of NH4Cl on the magnitude of the electrophoretic mobility of live and dead S.aureus (TiO2 as reference sample)
64
5.5.4 Effects of NH4Cl on the magnitude of the electrophoretic mobility of gram-positive and negative bacteria
66
5.6 Effects of different ionic strength of sodium chloride (NaCl) on surface charge of biological cells and colloidal particles
68
5.6.1 Effects of different ionic strengths of NaCl on the conductivity of electrolyte
69
5.6.2 Effects of NaCl on the magnitude of the electrophoretic mobility of live and dead E.coli (TiO2 as reference sample)
71
5.6.3 Effects of NaCl on the magnitude of the electrophoretic mobility of live and dead S.aureus (TiO2 as reference sample)
73
5.6.4 Effects of NaCl on the magnitude of the electrophoretic mobility of live E.coli and S.aureus
74
5.6.5 Conclusion of effects of NaCl 75
5.6.6 Donnan potential of live E.coli and S.aureus in various ionic strength of NaCl
75
5.6.7 Conclusion on curve fitting of Donnan potential 80
5.7 Summary on effects of the magnitude of the electrophoretic mobility in different chemical environment
80
Chapter 6 : Effects Of Temperature And Applied Field On Electrophoretic Mobility
6.0 Effects of temperature on the magnitude of the
electrophoretic mobility of colloidal particles and bacteria 82
6.1 Comparison of theoretical and experimental electrophoretic mobility of TiO2 and liposome
83
6.1.1 Comparison of zeta potential of colloidal particles at the range of temperatures
86
6.1.2 Comparison of the magnitude of the electrophoretic mobility of TiO2
and liposome at the range of temperature
88
6.1.3 Electrophoretic mobility live and dead Escherichia coli at a range of temperature (TiO2 as reference sample)
89
6.1.4 Electrophoretic mobility live and dead S.aureus at a range of temperature (TiO2 as reference sample)
93
6.1.5 Comparison of the magnitude of the electrophoretic mobility of gram positive and gram negative bacteria at range of temperatures
95
6.2 Effects of applied field on the magnitude of the electrophoretic mobility of colloidal particles and bacteria
98
6.2.1 Ohm’s plot for colloidal particles and bacteria at different applied field
101
6.2.2 Temperature changes against applied fields due to current increase
103
6.2.3 Conductivity against applied fields for colloidal particles and bacteria
105
6.2.4 Electrophoretic mobility of TiO2 and liposome at a range of applied field
107
6.2.5 Electrophoretic mobility of live and dead E.coli (TiO2 as reference sample)
110
6.2.6 Electrophoretic mobility of live and dead S.aureus (TiO2 as reference sample)
113
6.2.7 Electrophoretic mobility of live E.coli and S.aureus 116
6.3 Effects on different time interval between electrophoretic mobility measurements of live E.coli and S.aureus
117
6.4 Summary on the effects of different temperatures, applied field and time interval on surface charge
124
Chapter 7 : Polarization Of Bacteria And Colloidal Particles
7.0 Polarization of bacteria and colloidal particles 127
7.1 Clausius-Mossotti Approximation 129
7.2 Result and discussion on polarization of live cells and colloidal particles
131
7.3 Result and discussion on low frequency dependence Clausius-Mossotti formula
132
7.4 Summary of polarizability of bacteria and colloidal particle 134
Chapter 8 : Conclusion And Future Work
8.0 Discussion and conclusion 135
8.1 Future Works 138
References 140
List Of Publications 149
Appendix 150
LIST OF FIGURES
Figure
Page
2.1 (a) Gram-negative cell wall structure (b) Gram-positive cell wall structure
11
2.2 Simple structure of fluid mosaic model of cell membrane
15
2.3 The chemical structure of cholesterol
15
2.4 A simple structure of phospholipids
18
2.5 Bilayer phospholipid structure of liposome
18
3.1 Side view illustration of negatively charged particle is surrounded by opposite charges on its surface and forms the electrical double layer.
24
3.2 Illustration of the Hückel and Smoluchowski limits of the Henry’s function where a, is the particle radius and к-1 is the Debye length or double layer thickness
29
4.1 The folded capillary cell for electrophoretic mobility measurement which designed by Malvern’s Zetasizer Nano Series
35
4.2 The occurring of the light scatters at angle of 17o and produces the intensity of scattered light.
36
4.3 Two waves of slightly different frequencies (Base wave and Frequency Shifted) along with the summation of the two
38
4.4 Stationary layer where two laser beams cross is free from electroosmotic effect.
39
4.5 The arrangement of the components for zeta potential measurement with Zetasizer Nano series instrument
43
4.6 The components show in Figure 4.5 except computer was compacted in this plastic box with the mentioned size
43
5.1 (a) The micrograph of S.aureus. (b) The micrograph of E.coli O157:H7.
45
5.2 Diameter sizes of the liposome and TiO2
46
5.3 (a) Micrograph of TiO2 (b) Micrograph of caprate-capric acid liposome
46
5.4 Comparison of the magnitude of the magnitude of the electrophoretic mobility live and dead E.coli (TiO2 as reference sample) at various pH (Polynomial least square analysis is shown)
52
5.5 Comparison of the magnitude of the electrophoretic mobility live and dead S.aureus (TiO2 as reference sample) at various pH (Polynomial least square analysis is shown)
55
5.6 Conductivity, Λ of TiO2, live and dead E.coli and S.aureus in different concentrations, c of NH4Cl
61
5.7 The magnitude of the electrophoretic mobility of live and dead E.coli (TiO2 as reference sample) versus the reciprocal of the square root of the ionic strength of NH4Cl. The dots with error bars are experimental points, the best-fitted continuous and dashed lines were plotted with second order of polynomial least square analysis
62
5.8 The magnitude of the electrophoretic mobility of TiO2, live and dead S.aureus versus the reciprocal of the square root of the ionic strength of NH4Cl. The dots with error bars are experimental points, the best-fit continuous and dashed lines were plotted with using second order of polynomial least square analysis
65
5.9 Molar conductivity, Λ of TiO2, live and dead E.coli and S.aureus in different concentrations, c of NaCl
70
5.10 The magnitude of the electrophoretic mobility of live and dead E.coli (TiO2 as reference sample) versus the reciprocal of the square root of ionic strength of NaCl. The dots with error bar are the experimental points, the lines and dash lines of best fit are plotted with second order of polynomial least square analysis.
71
5.11 The magnitude of the electrophoretic mobility of live and dead S.aureus (TiO2 as reference sample) versus the reciprocal of the square root for ionic strengths NaCl. Dots with error bars are experimental points. Solid and dashed lines of best fit are plotted with second order polynomial least squares analysis
73
5.12 The magnitude of the electrophoretic mobility of ionic strength (NaCl) curve for E.Coli at pH 7. Open circles indicate numerical values and the filled triangles demonstrate experimentally measured the electrophoretic mobility with the Zetasizer instrument
79
5.13 The magnitude of the electrophoretic mobility of ionic strength (NaCl) curve for S.aureus at pH 7. Open circles indicate numerical values and filled triangles demonstrate experimentally measured the electrophoretic mobility with the Zetasizer instrument
79
6.1 Hückel–Onsager and Helmholtz-Smoluchowski predicted electrophoretic mobility values compared with experimental values of TiO2 in a range of temperatures
84
6.2 Hückel–Onsager and Helmholtz-Smoluchowski predicted electrophoretic mobility values compared with experimental values of liposome in a range of temperatures
85
6.3 The comparison of zeta potential between liposome and TiO2.
86
6.4 Actual and predicted electrophoretic mobility of TiO2 and liposome at various temperatures and the predicted curve was based on the Helmholtz-Smoluchowski equation
88
6.5 Effects of temperature (20oC-55oC) on electrophoretic mobility of TiO2, live and dead E.coli (Electrophoretic mobility of TiO2 measured is help to guide the trend as temperature increased)
90
6.6 Zeta potential of live and dead E.coli corresponded to the magnitude of electrophoretic mobility that obtained from measurements.
92
6.7 Effects of temperature (20oC-55oC) on electrophoretic mobility of TiO2, live and dead S.aureus (Electrophoretic mobility of TiO2 measured is help to guide the trend as temperature increased)
93
6.8 Zeta potential of live and dead S.aureus, which corresponded to the magnitude of electrophoretic mobility.
94
6.9 Effects of temperature (20oC-55oC) on electrophoretic mobility of E.coli and S.aureus
96
6.10 Zeta potential measured of E.coli and S.aureus corresponded to the magnitude of the electrophoretic mobility.
96
6.11 Ohm’s plot for colloidal particles and bacteria in varied applied field
102
6.12 Temperature changes when applied field increase for colloidal particles and bacteria
104
6.13 Conductivity of colloidal particles, live and dead E.coli and S.aureus against applied field
106
6.14 Electrophoretic mobility of TiO2 and liposome for both result from experimental and estimation from calculation
108
6.15 Zeta potential which corresponded to the magnitude of the magnitude of the electrophoretic mobility of TiO2 and liposome
109
6.16 Actual and predicted electrophoretic mobility of live and dead E.coli (Electrophoretic mobility of TiO2 as reference)
111
6.17 Zeta potential which corresponded to the magnitude of the electrophoretic mobility of live and dead E.coli
112
6.18 Experimental and predicted electrophoretic mobility of live and dead S.aureus (Electrophoretic mobility of TiO2 as a reference)
114
6.19 Zeta potential which corresponded to the magnitude of the electrophoretic mobility of live and dead S.aureus
115
6.20 Electrophoretic mobility and the corresponded zeta potential of live E.coli and S.aureus
116
6.21 (a) Zeta potential of live E.coli and S.aureus for time interval at 1s (b) Zeta potential of live E.coli and S.aureus for time interval at 3s
119 119
6.21 (c) Zeta potential of live E.coli and S.aureus for time interval at 5s. (d) Zeta potential of live E.coli and S.aureus for time interval at 7s.
120 120
6.22 (a) Zeta potential of live E.coli and S.aureus for time interval at 10s (b) Zeta potential of live E.coli and S.aureus for time interval at 60s
122 122
6.22 (c) Zeta potential of live E.coli and S.aureus for time interval at 80s (d) Zeta potential of live E.coli and S.aureus for time interval at 120s
123 123
7.1 Predicted conductivity of liposome, live E.coli and S.aureus with frequency less than 1.0 Hz
133
7.2 Particle permittivity and compare to the ratio of particle permittivity over the electrolyte permittivity were estimated in higher frequency range.
133
LIST OF TABLES
Figure
Page
5.1 The magnitude of the electrophoretic mobility of live E.coli and S.aureus after number of wash.
47
5.2 The magnitude of the electrophoretic mobility of TiO2, live, dead E.coli and S.aureus suspended in distilled water.
48
6.1 Temperature coefficients, α is calculated for colloidal particles and bacteria.
105
7.1 Polarization of bacteria and colloidal particles.
131
LIST OF ABBREVIATIONS
TiO2 - Titanium dioxide S.aureus - Staphylococcus aureus E.coli - Escherichia coli
CHAPTER 1
Electrophoretic Studies of Surface Charge on Unicellular Bacteria
1.0 Introduction Surface charge properties are important in understanding how microorganisms
behave and interact with their environment. Bacteria tend to adhere to a liquid
environment rich in nutrients, the instances tears , saliva [3] , human serum, food [4], or
wastewater. The adhesion of bacteria to different environments depends on the
charges of the surface. Besides, surface charge properties provide information about cell
surface compositions [5, 6], isoelectric points , rates of nutrient uptake, pH and ionic
strength [7, 8].
Bacteria are living cellular microorganisms that are resistant to changes to its
surface charge in order to survive in various environments. Consequently, the surface
charge of a colloidal suspension is needed verify and complement surface charge
experimental results. A colloidal particle such as liposome, which is comprised of a
simple cell membrane structure, has often been used as a model to encapsulate drugs for
delivery into the human body [9]. Charged colloids or cells suspend in dispersant
attempt to achieve electroneutrality by attracting oppositely-charge ions. This charge
can be measured with migration rates of charged particles when placed in an electric
field. The electrophoretic mobility of charged particles can be obtained and the zeta
potential, which measures surface charge potential, can hence be estimated at a given
distance from charged particles [10].
There are two major categories of bacterial cells, namely, gram-negative and
gram-positive bacteria. In gram-positive bacterial cell walls, ionizable functional groups
are associated with peptidoglycan and secondary polymers such as teichoic or
teichuronic acids [11]. Carboxyl functional groups attached to the unlinked peptide
crosslinks of peptidoglycan and phosphoryl groups associated with the teichoic acids
can deprotonate to form negative charges which subsequently adhere to
positively-charged materials [12]. On the other hand, gram-negative bacterial cells
comprise a thinner cell wall peptidoglycan layer and lipopolysaccharides [13] which are
associated with more protein integrity than gram-positive bacteria in cell membrane [14]
and these forms the negatively-charge bacteria. The negative surface charge of bacteria
can be changed when suspend in different chemical conditions. The neutralization of
opposite ions on surface charge in the suspension medium can cause flocculation
between cells [15]. Consequently, studies of bacterial surface charge properties have
shed more light on the industry of biochemical interactions.
Studies on cell surface charge reveal the mechanism of the cellular interactions
and how cells respond to environmental changes. Such a research has been done on
various types of living and non-living cells. Living cells that are of most interest for
their electrochemical changes are microbes, human cells [16] and plant cells [17]. On
the other hand, non-living cells consist of colloidal particles which are studied for their
surface charges in order to improve particle stability and flocculation [18, 19] . For
years, studies on biochemical interaction, which is related to bacteria surface charge,
have improved drugs usage of drugs to cure bacterial infection. Most research has
focused on cellular responses drugs and changes of its surface charge. This has
enhanced the importance of studies on cell membrane surface charge [20].
1.1 Literature review
In recent years, surface charge studies have created an interest in various cells.
Gram-positive and gram-negative bacteria have been widely been used to measure
electrophoretic mobility. Studies of bacterial surface charge properties facilitate the
development of biomedical applications, which focus on adhesion and colonization of
bacteria in biomedical devices. For instance, such development include adhesion of
bacteria to contact lenses, dental implants, urethral stents and prosthesis [21]. This is
important because aquatic environments such as tears, saliva, sewage systems, human
serum, and wastes water are enriched with nutrients which fertilize the growth of
bacteria. Adhesion of bacteria to bio-devices can be avoided by coating appropriate
materials on the surface of the devices, depending on the bacterial surface charge.
Related studies on surface charge have also reported microbial response to external
conditions such as heat, chemical stress, nutrients deficiency and osmotic factors [22].
Hydrophobicity and electrophoretic mobility is a measure to predict the initial steps of
bacterial adhesion. Growth rate and conditions also influence cell surface characteristics
that determine adhesion [23].
Microbial behaviour is commonly known to be controlled by the
physico-chemical properties of the cell membrane [24, 25]. It is important to carry out
quantitative measurements on the surface charge of both harmful and probiotic bacteria.
Study on the surface charges of various harmful bacteria has led to the identification
chemicals which suit to inactivate or denature harmful bacteria. This is important to
ensure that water from the sewage pond is free from harmful bacteria before flowing
back to the river [26, 27]. Staphylococcus aureus have been found to constitute a large
percentage of infections associated with implanted biomedical devices. This discovery
has led to the designing of a novel implant material based on the concept of repulsive
forces on biofilm formation, thereby reducing the growth of Staphylococcus aureus
[11].
Study on surface charge properties has also contributed to agriculture
development. Investigations have been carried out on cell surface charge and
hydrophobicity in several strains of Azospirillum, which has been associated with the
better plant growth [28]. This has provided greater insight into the external changes of
bacterial onto plant root surfaces. The effects of monascus pigment on bacterial
eletrophoretic mobility, cell absorption of monascus pigment and the antibacterial
activities of pigments have been investigated [29]. In the medical field, research has
reported on electrical properties of normal cells interaction depends on surface charge,
thereby the changes of surface charge of a cell will influence the adjacent cells and
affects amount of new cells production daily. If the surface charges of cells do not
change according to environment, these group of cells would turn into cancerous cells
[30, 31]. Surface charge study has contributed to the detail understanding of cell
communications and, hence, can improve on cancer treatement.
The surface charge of bacteria can be changed by different conditions, such as
pH and ionic strength. In a study of the electrophoretic mobility of wild-type
Escherichia coli and its O157:H7 strain across a range of pH and ionic strength,
decrease in pH increased the electrophoretic mobility of both strains [10]. In addition,
Joule heating in an electrophoretic system containing various salt concentrations greatly
affected electrolyte electrophoretic mobility [32]. Another study of reported that the
surface charge decreased as the hydrophobicity of gram-positive bacteria
Staphylococcus aureus increased in adherence to hydrocarbon by measured the change
in electrophoretic mobility [33].
A study on surface charge was done on streptomycin to Escherichia coli and
Staphylococcus aureus. It was found that chemical streptomycin reduced the net
negative charge of Escherichia coli but had no effect on Staphylococcus aureus [34].
Another surface charge research was on the effects of milk from bovine mastitis on the
surface charge of Staphylococcus aureus reported that the hydrophobicity of bacteria
was reduced and heat treatment of a certain range did not significantly change surface
charge properties [35]. Measurements of electrophoretic mobility in Escherichia coli
and Staphylococcus aureus has found differences in the surface properties of
gram-negative and gram-positive bacteria [36].
Much research on the electrophoretic mobility of colloidal suspension has been
done [37, 38]. Physicochemical interactions are essential in determining the stability of
colloidal media such as emulsion or food metrices. The same type of interaction is
involved in the attachment processes of microorganisms through their surface properties
to the interface of the emulsion or food [39]. Colloidal particles such as liposome are
well established as carriers of antimicrobial and anticancer agents. The liposome is a
simple model of biological cell membranes that has been used widely. Liposome
forming synthetic amphiphile was evaluated in a few types of bacteria. This was to
investigate the interactions between cationic liposomes and bacteria and also the
physical-chemistry of bactericidal action [40].
Moving on to another colloidal particle, titanium dioxide (TiO2) is commonly
used in many industrial applications. The white pigment powder possesses a high
refractive index, leading to a wide usage of TiO2 as a catalyst support [41, 42],
semiconductor photocatalyst [43], antimicrobial coating [44], oxygen sensors and as UV
filters for cosmetic products and paints [45]. These applications are possible due to the
surface properties of TiO2 which remain uncertain when affected by the change in pH,
ionic strength and temperature [46] . Experimental studies have characterized the
colloidal stability of TiO2 and water interfaces in the presence of several electrolytes
and at different pH ranges [47]. The colloidal stability of TiO2 in different concentration
ranges of dispersant of polyacrylic-based deflocculant and citric acid was studied and it
was found that the zeta potential of TiO was affected by sonication time [48]. 2
1.2 Scopes of the Study
Two strains of bacteria, (E.coli) Escherichia coli and (S.aureus) Staphylococcus
aureus were selected for this study. Two types of colloidal particles were chosen to be
control sample to biological cells, namely liposome and titanium dioxide (TiO2). One of
the assumptions in this study was that samples were in spherical, including E.coli which
is cylinderical. The colloidal particles and bacteria were investigated for their negative
surface charges with electrophoresis integrated with Laser Doppler Velocimetry
electrophoresis system. Colloidal particles selected were TiO2 and liposome. The
bacterial strains in this study consisted of gram-positive S.aureus and gram-negative
E.coli. Both live and dead bacteria were investigated. It is very convenient to study the
surface electrical properties of microparticles. Conventionally, electrophoresis is
supplied with a direct current which causes the accumulation of air bubbles at the
electrodes as the operation time is prolonged. Consequently, the electrophoretic system
used here has been shown modified by using an alternating current. This technology
also includes the technique of phase analysis light scattering (PALS) which can measure
the velocity of particles with very low conductivity in solution [1] .
The study is devoted to the understanding of the surface charges of unicellular
organisms by using Laser Doppler Electrophoresis with phase analysis light scattering
techniques. This study investigated the electrophoretic mobility and analysed a variety
of cells and colloids namely, TiO2, liposome, live and dead E.coli and S.aureus. An
investigation of the changes in the electrophoretic mobility unicellular organisms in
various conditions is discussed. The different conditions consisted of various ranges of
temperature, applied voltages, buffer pH, various time intervals between measurements,
and different concentrations of sodium chloride and ammonium chloride. The Donnan
potential was simulated to compare the electrophoretic mobility of live bacteria. The
theoretical bases of surface charges of unicellular cells and colloids are discussed.
1.3 Outline of the Thesis
The thesis comprises seven chapters. The following is an outline of the scope for
each chapter.
Chapter Two introduces the essentials of each of selected sample. The negative
surface charge of samples in suspension is described.
Chapter Three discusses the theory of the surface charges which is to introduce the
relation between the zeta potential and electrophoretic mobility.
Chapter Four is about the principle of the Laser Doppler Electrophoresis system
which is used to measure the electrophoretic mobility of unicellular organisms. In this
chapter, the methodology and preparation of the materials is explained in detail.
In Chapter Five, the results and discussion of the effects of temperatures, applied
fields and different time intervals between measurements on the electrophoretic
mobility of colloidal particle and bacteria are presented.
Chapter Six presents the electrophoretic mobility measurements of samples with
numbers of cells centrifugations, distilled water suspension, ranges of pH, and different
concentrations of NaCl and NH Cl. 4
Chapter Seven discusses the polarization of the live cells.
Chapter Eight comprises an overall conclusion of the results of this study.
Suggestions and discussions concerning the improvement of the methodology employed
in studying the surface charges of unicellular organisms are carried out. Future
investigation is proposed.
CHAPTER 2
Characterization of Surface Charge Bacteria
2.0 Introduction to gram-positive and negative bacteria
Most of microorganisms are unicellular. A simple single cell contains all the
necessary structures to manage internal physiology and deal with the external
environment. Microorganisms, which include bacteria, have brought advantages and
disadvantages to the environment. For instance, plant legumes rely on certain bacteria to
convert nitrogen gas to ammonia which is an essential plant nutrient [49, 50] . On the
other hand, S.aureus is a major human pathogen that can infect almost every tissue in
the body [51]. A study reported the effects of electrophoresis on the ionic toothbrush in
which the negative ion on the toothbrush originated from S.aureus. As a result, this is
important to develop a toothbrush for hypersensitive teeth [52].
Microorganisms on earth have evolved different cellular structures to survive in
different conditions. There are two major categories of microorganisms, namely
prokaryotic and eukaryotic microbes. Eukaryotes are distinguished by the present of a
nucleus, vacuole, mitochondria and a more complex cellular system, whereas
prokaryotes do not have a nucleus and simpler structures. Both types of microbes
consist of cell membranes, cytoplasm and ribosomes for protein synthesis. Besides, the
eukaryote is about ten times bigger than the prokaryote. The diversity of eukaryotic
microbes is lesser compared to prokaryotic microbes. This is because prokaryotic
microbes have existed longer on earth in order to diversify into different species [53,
43] .
Two species of prokaryotic bacteria were selected in this investigation, namely,
Escherichia coli (E.coli) type O157:H7 and Staphylococcus aureus (S.aureus). These
two species of bacteria are from two different classifications. E.coli is gram-negative,
[54, 55, 10] whereas S.aureus is gram-positive [10, 11, 12]. The main difference
between gram-positive and -negative bacteria is their cell wall structure. The
gram-positive bacterial cell wall is made up of a thick sheath of peptidoglycan and
tightly bound with acidic polysaccharides. The cell wall of gram-positive bacteria
contains teichoic acid which is a ribitol polymer or glycerol and phosphate embedded in
the peptidoglycan sheath.
The gram-negative bacterial cell wall is much more complex. The cell wall of
gram-negative bacteria consists of a rigid peptidoglycan layer that is much thinner than
that of gram-positive bacteria. The cell wall of gram-negative bacteria is also overlaid
with an outer membrane, which is a gel-like periplasm in-between the cytoplasmic
membrane and the outer membrane. The phospholipids of the cell wall of
gram-negative bacteria contain a toxic layer of various proteins, lipoproteins and
lipopolysaccharides (LPS) [56, 57, 58, 59]. Thus, the major difference between
gram-negative and -positive bacteria is the thickness of their peptidoglycan layer and
not so much the cellular chemical composition as depicted in Figure 2.1 (a) and (b)[60] .
Figure 2.1 (a) Gram-negative cell wall structure. (b) Gram-positive cell
Peptidoglycan
Cytoplasm
Cytoplasmic membrane
Cytoplasm
PeptidoglycanOuter membrane
Cytoplasmic membrane
2.1 Escherichia coli and Staphylococcus aureus for surface charge study
E.coli O157:H7 is a well-known gram-negative rod-shaped bacterium. The letter
"O" in the name refers to the somatic antigen number, whereas "H" refers to the flagella
antigen [61]. E.coli has flagella for motility. Flagella are long appendages which rotate
by means of a "motor" located just under the cytoplasmic membrane. Bacteria may have
one, few, or many flagella at different positions on the cell. E.coli infections among
humans can lead to bloody diarrheoa, and occasionally to kidney failure, especially in
young children and the elderly. The outbreaks of E.coli O157:H7 infection is frequently
associated with eating undercooked beef [62], mutton and lamb. An investigation in the
USA even found that one of the sources of infection was an unpasteurised apple-juice
product [63]. E.coli O157:H7 are generally more tolerant of adverse environmental
conditions than many non-pathogenic E.coli strains [64]. Studies have that the minimum
pH for E.coli growth is 4.0 to 4.5 [65] and this is also dependent on the interaction
between pH and other growth parameters such as temperature. E.coli can be
conducively cultured from 21oC-37oC [66]. Researchers have reported heat inactivation
of E.coli O157:H7 at 55, 58 and 60 oC, with a simultaneous application of pressure. For
the latter treatment, cells inactive for 10 days but active after 15 days [67].
The second species of bacteria in this study was S.aureus, which is gram-positve
and shaped as a cluster-forming coccus [68].The Staphylococcus genus includes thirty
one species and most of them are harmless [69]. Their cell walls are composed of
murein [70], teichoic acids [71] and surface proteins [72]. The normal temperature
used to culture is from 10o oC [69] to 42 C [73]. The bacterial colony appears golden
yellow on agar. S.aureus is a non-motile and non-spore forming facultative anaerobe. It
is normally found on human nasal passages, skin and mucous membranes [74].
However, it is categorized as a pathogen to humans as it can cause a wide range of
infections, as well as food-poisoning and toxic shock syndrome. On S.aureus adhering
to food studies have shown that a culturing temperature [75] of 52.5 to 54oC decreased
bacterial count [58]. Another study reported that S.aureus adhered better at 12oC on
food than at 30 oC, over 4h to 6h of incubation [76]. A variety of methods have been
developed to prevent infection by S.aureus [77, 52]. Nanoparticles of silver and
copper are widely used for antimicrobial studies, for which S.aureus and E.coli are
choices of investigation [78]. There have also been studies on the effects of high
pressure on the kinetics of inactivation in S.aureus. Since S.aureus is a disease-causing
pathogen. Studies of the characterisation of S.aureus should be broader.
2.2 Colloidal particles as a model of biological cells
The electrophoretic mobility of two colloidal particles used in this study, namely,
titanium dioxide (TiO2) and liposome, were compared with E.coli and S.aureus. Both
are well-known colloidal particles used in studies of surface charge properties. Since
TiO2, a white pigment powder, has a high refractive index, this has led to its wide usage
as a catalyst support, semiconductor photocatalyst in paints, antimicrobial coating [79],
oxygen sensors and as UV filters for cosmetic products [80]. The extensive usage of
TiO2 in consumer products has attracted interest among many researchers to investigate
the effects of TiO on the human body. Moreover, it has been reported that TiO2 2 could
damage biological targets through oxidative stress [81]. Another study demonstrated
that when used as photocatalyst, TiO2 generated reactive species which were damaging
to cell structure [82]. Hence, studies of the electrophoretic mobility surface charge of
TiO2 in varied conditions would not only help in the discovery of further applications in
different fields, but also in preventing potential hazards to living beings.
The liposome was the second colloidal particle used in this study, whose surface
charge properties were compared with that of bacteria. A liposome is formed from
self-assembling phospholipid molecules into bilayer structures that are similar to the
basic structure of a biological membrane which consists of a phospholipid bilayer
matrix [83]. This special characteristic of liposome has supported applications in drug
deliveries [84]. In addition, liposome also helps in improving the medical treatments for
various health problems. For instance, there is research on the application of boron-
entrapped stealth liposome to boron neutron capture (BNC) therapy for cancer treatment
[85]. Consequently, it is essential to investigate the effects of liposome composition on
adsorption and transport processes based on cell membrane charges [86]. Differences in
electrophoretic mobility have been utilized to monitor the effects of the compositional
variation in membrane fluidity and permeability on the apparent surface charges of
liposome [87].
2.3 Negative surface charge of bacteria and colloidal particles
The bilayer membrane of the biological cell which separates the interior from the
surrounding fluids is largely composed of phospholipids. This layer incorporates many
other components, such as cholesterol, which contributes to its structural integrity. The
protein channels that permit the transport of various kinds of chemical species in and
out of the cell are also important components of cell membranes. A simplified structure
of a cell membrane is shown in Figure 2.2.
Integral Protein
Glycolipids
Cholesterol
Phospholipid
Integral Protein
Peripheral Protein
Figure 2.2 Simple structure of fluid mosaic model of cell membrane.
The phosphate head of phospholipids may have additional polar groups such as
choline, ethanolamine, serine and inositol; all of which increase cell hydrophilicity.
Besides, the cholesterol (steroid) molecular in Figure 2.3 has a polar hydroxyl group
that points towards the surface of the cell [88].
HO
H
H
H
Figure 2.3. The chemical structure of cholesterol.
The membrane is fluid-like and its phospholipids exhibit rapid lateral movement,
but rare flip-flop movement. The diffusion coefficient of lipids in a variety of
membranes is about 1 μm2 s-1. A phospholipid molecule diffuses an average distance of
2 μm in 1 s [89]. The membrane bilayer consists of protein channels formed by integral
proteins that allow ions to diffuse across the membrane. Different cells have different
permeability to these ions. The diameter of the channel and the polar groups on the
protein subunits forming channel walls determine the permeability of the channels by
various ions and molecules [90].
Membrane potential is the separation of electric charges across a membrane, similar
to the voltage across a membrane. The separation of charges influences the movement
of ions across the membrane. This is due to the unequal distribution of cations and
anions across the plasma membrane. Consequently, all cells have voltages across their
plasma membranes. The membrane potential of live cells ranges from about -50 mV to
-200 mV [91] . The negative sign of the value indicates that the cell interior is
negatively charged with respect to the exterior. This affects the traffic of charged
substances across the membrane, in that cations diffuse into the cell while anoions
diffuse out due to electrostatic attraction. The factors that contribute to the net negative
charge of a cell’s membrane potential includes the negatively-charged proteins in the
cell interior and the sodium-potassium pump which translocates 3 ion Na+ out for every
2 K+ that enters the cell yielding a net loss of one positive charge per cycle.
Consequently, cells carry a negative surface charge [92] .
The selected colloidal particles employ in this study to compare with the surface
charge of bacteria was titanium dioxide, TiO2 and liposome. TiO2 is a solid metal oxide
particle. The surface charge of this metal oxide arises from the protonation and
deprotonation of the metal hydroxyl group. The oxide group in the metal oxide is
stronger with negative bonds although the particle is neutral. Hence, the particles tend to
bind with the hydrogen of water molecules, causing the oxygen molecules to face
outwards towards the surroundings. Consequently, TiO2 is negatively-charge when
suspended.
_2 OMOHMOHM −−↔−−↔−− +
In general, increasing the pH will deprotonate the surface, causing a negative
surface charge, whereas lowering the pH gives a positive surface charge [93].
Phospholipids are the main constituents of cell membranes. A basic phospholipid
has a hydrophilic (polar) head and two hydrophobic tails. Phospholipid diversity is
based on the difference in the fatty acids and in the groups attached to the phosphate
group of the head [92]. Figure 2.4 shows the simple structure of a phospholipid. The
phospholipid molecules can move about in their half of the bilayer, but there is a
significant energy barrier preventing migration to the other side of the bilayer.
Liposome in this study was formed from capric acid which is a common saturated fatty
acid. When phospholipids are added to water, single layer of phospholipids would form
on the water surface. The hydrophilic head will be exposed to water due to the polar
head groups, while the hydrophobic alkyl chains form a non polar tail are excluded from
water. With the help of sonication, a micelle is formed. Adding more phospholipids to
water will form a bilayer of phospholipids of spherical shape called a liposome.
However, a liposome is different from a micelle, in that a liposome has both an aqueous
interior and exterior. Since the hydrophilic head faces the exterior aqueous environment,
a liposome carries a negative surface charge. This is depicted in Figure 2.5.
Hydrophilic (polar)
Hydrophobic tails (non-polar)
Figure 2.4 A simple structure of phospholipid.
Aqueous interior
Aqueous exterior
Figure 2.5 Bilayer phospholipid structure of liposome.
Negative surface charge
2.4 Summary of surface charge
Samples in this study are negatively-charged. The gram-positive and -negative
bacterium which is S.aureus and E.coli respectively are biological cells that have
different cell wall thickness and protein composition. The similar characteristic of both
bacteria is carrying negative surface charge. The factors that contribute to a negative
surface charge of biological cell membranes are listed as follows:
• Cholesterol (ending with a hydroxyl group).
-• Choline, ethanolamine, serine, carboxylic acid group (COOH ) and
inositol, all of these components are having a phosphate group (PO4-).
• A tendency to transport cations into the cell and anions out of the cell.
TiO2, whose negative surface charge is compared with biological cells. It carries
negative charges at higher pH but more positive charges at lower pH, depending on
protonation and deprotonation effects in various suspensions. Lastly, liposome is
-formed from phospholipids which possess carboxylic acid group (COOH ) in the
hydrophilic (head) that forms a negatively-charged surface.
CHAPTER 3
An Approach to Surface Charge and Electrical Double Layer
3.0 Introduction to Surface Charge and Electrical Double Layer
Mechanism of a cell or particle acquires charges when exposes to liquid was
discussed in this study. Subsequently, it is essential to understand the relationship
between the ions on the surface and those exist in the solution. Hence, some basic
concepts concerning the origin and qualitative structure of the ions atmosphere that
develops in the vicinity of a charged surface exposed to a solution containing ions are
discussed.
3.1 Origin of Charges at a Surface
A surface immerses in a liquid can acquire charges in a number of ways. One of
the common charges acquiring process of a surface is preferential adsorption of an ion
from a solution on an initially uncharged surface. This is the “charging” mechanism for
TiO , which was used in this study. TiO2 2 is a metal oxide particle, which the surface
charges of this metal oxide arises from the protonation and deprotonation of the metal
hydroxyl group. The oxide groups in the metal oxide more influencing although the
particle is neutral. Hence, the particles tend to bind with the hydrogen of water
molecules, causing the oxygen molecules to face outwards towards the surroundings.
Hence, TiO is negatively-charge when suspended. 2
Another possible mechanism is ionization and dissociation of a surface group,
for instance, the dissociation of a proton from a carboxylic group, which leaves the
surface with a negative charge. In this research, liposome as a cell like particle was
formed from capric acid. The hydrophilic head of liposome exposes to water due to the
polar head groups, while the hydrophobic alkyl chains form a non polar tail are
excluded from water. Since the hydrophilic head faces the exterior aqueous environment,
a liposome carries a negative surface charge.
Gram-positive and –negative bacteria are carrying negative surface charge [94,
95] , which influencing by different pH solution. There is study reported that between
pH 5 and 7 suspension medium for most of bacteria strains are negatively-charged [94].
In higer acidic environment, bacteria might be more electropositive.
3.2 Introduction to Electric Double Layer
Surface charge of bacteria cells is measured with electrophoresis due to
development of net charge at the particle surface affects the distribution of ions in the
surrounding interfacial region. This process increases concentration of counterions (ions
of opposite charge to that of the particle) close to the surface. Consequently, an
electrical double layer exists around each particle which describes the variation of
electric potential near a surface, and has a large bearing on the behaviour of colloids,
cells and other surfaces in contact with solutions [96].
The first model named 'electrical double layer' was introduced by Helmholtz in year
1850 [97]. This model divides electrical double layer into two parts, compact part
adjacent to the surface and a diffuse part. The compact part near the surface of a
charged particle consists of oppositely-charged ions [96] and immobile. This is caused
by the strong electrostatic forces of charged particle and oppositely charged ions. At
diffuse part, oppositely charged ions towards charged particles are weaker in
electrostatic force due to the further distance from the surface of the charged particle,
thus the counterions at this part are mobile and the co-ions around the layer tends to
repel. This part is also called slipping plane.
Ions around the charged particles at the compact part can be defined with
Helmholtz planes in which the inner plane represents the closest approach of nonhyrated
ions to the surface. A nonelestrostatic adsorption may occur at a distance of
approximately 0.1 to 0.2 nm from the surface. Meanwhile, the outer Helmholtz plane is
described as the closest approach of hydrated ions, which is about 0.6 nm from the
surface of charged particles [93]. The relative dielectric permittivity of region between
surface charged particle and inner Helmholtz plane are called Stern layer, shown in
Figure 3.1. Counterions distribute at the outer Helmholtz layer to end of the diffuse
layer creates potential difference, which the outer Helmholtz layer determines the
beginning of diffuse layer. This potential difference names diffuse layer potential.
Counterions surround at the surface charged particles are highly concentrated near
the surface but gradually decreases with distance until reaches equilibrium with
counterions concentration in the solution. Initially, negative ions in the solution are far
away from the neighborhood of negatively-charged particle. However, the concentration
of these negative ions will gradually increase with distance as the repulsive force of the
particle is screened out by positive ions until the equilibrium is again reached [98].
Figure 3.1 shows the ions distribution around a negative charged particle.
The Helmholtz theory does not adequately explain all the features of diffuse double
layer, which hypothesized the rigid layers of opposite charges [99]. This model was
further improved by Gouy and Chapman’s model, who suggested that interfacial
potential at the charged surface could be attributed to the presence of a number of ions
of given sign attached to its surface, and to an equal number of ions of opposite charge
in the solution. In other words, counter ions are not rigidly held, but tend to diffuse
into the liquid phase until the counter potential set up by their departure restricts this
tendency. The kinetic energy of the counter ions will, in part, affect the thickness of
the resulting diffuse double layer. Gouy-Chapman developed theories of this is called
diffuse double layer in which the change in concentration of the counter ions near a
charged surface follows the Boltzmann distribution [100].
(3.1)
)/( kTzFocc −=
where co is denoted as bulk concentration, z is charge on the ion, F is Faraday constant,
k is derived as Boltzmann constant and T is the absolute temperature in Kelvin.
Inner Helmholtz layer
Outer Helmholtz layer
Pote
ntia
l diff
eren
ce, ψ
Zeta potential, ζ
κ-1Distance
Stern layer Diffuse layer
Figure 3.1. Side view illustration of negatively charged particle is surrounded by opposite charges on its surface and forms the electrical double layer.
3.3 Introduction to Zeta Potential
The diffuse layer potential, ψd is identified as zeta potential, ξ. It is fully defined
by the nature of the surface with charges which usually determined by pH, the
electrolyte concentration in the solution and the solvent [101]. For any interfaces of
charged particles in fixed parameters, zeta potential is a well defined surface charge
property. However, the zeta potential for the same system might be varied due to the
influential of minor impurities in solution [101].
Zeta potential is generally determined by electrokinetic techniques, for instance,
electrophoresis. It is also an estimation value from measurement of the electrophoretic
mobility with the unit of milivolts. The value of zeta potential is important to be used
for controlling the stability of colloidal suspension [102]. This is because of the
repulsive forces between the colloid particles can be maximized by monitoring the value
of zeta potential. The strong repulsive force of colloidal particles in suspension is
essential to keep each particle discrete and prevent them from aggregating into larger,
faster settling and later causes agglomeration. On the other hand, estimation of zeta
potential is important for biological cell. The zeta potential measured from bacteria in
suspension can help to gain better information on surface charge properties of the strain
and enable the estimation of adhesion properties of the bacteria onto various types of
materials [103] .
3.3.1 Electrokinetic Phenomenon: Electrophoresis
Electrophoresis was found by Reuss in 1809 when he discovered the clay particles
dispersed in water migrate under the influence of electric field [104, 2]. Latterly,
electrophoresis is defined as motion of dispersed particles relative to a fluid when
responses to the applied electric field.
The velocity of charged particles can be determined with electrophoresis. There are
two forces acting on the charged particles in a suspension. Thus, the charged particles
migrate at a terminal velocity when equilibrium between the forces is reached. The
existing forces are electrostatic force (FE=QE, where the Q is charge and E is the
electric field) acting on charged particle which suspends in liquid or solution when
voltage is applied. Another force acts oppositely is Stokes drag or friction force of the
particle (Fs= 6πηav, where a is the radius of the particle and v is the velocity of the
charged particle). Both forces are equal and exist simultaneously when the particle
starts to move.
However, most of the electrostatic force is applied to the counterions at the
diffuse layer of charged particle. Part of this electrostatic force transfers to the particle
surface through the shear of the viscosity of water. Consequently the electrophoretic
mobility is defined as [105]
aQ
Ev
e πημ
6=≡ (3.2)
where μe is electrophoretic mobility, v is the velocity of the particle moves to the
electrode and E is the strength of electric field. Take the zeta potential to be equal to the
surface potential, ψs. The Coulomb’s law gives Q/εa=ζ ≈ ψ = (recall that the ε=4πε εs o r).
Equation 3.2 turns into 3.3 and this formula is known as Hückel–Onsager equation.
πηεςμ
6=≡
Ev
e (3.3)
The formula was further improved to relate it to zeta potential estimation. According to
Smoluchowski’s equation, the electrophoretic velocity, ve can be defined as below
[101],
Ev ore η
ςεε= (3.4)
where the ε is relative permittivity of the electrolyte solution, εr o the electric permittivity
of vacuum, E is the applied electric field, ζ is zeta potential and η is defined as viscosity
of the solution and the this gives the electrophoretic mobility, μe.
ηςεε
μ ore = (3.5)
Equation 3.4 is known as the Helmholtz-Smoluchowski’s equation for electrophoresis
measurement. However, there are certain conditions for the electrophoretic mobility to
be converted to zeta potential. First, κa of the suspension must be determined as there
are two different equations for κa>1 and κa<1, which the κ is the Debye length and a is
the radius of the particle.
The electrophoretic mobility, μe is obtained for different range of indifferent
concentrations of solution, if electrophoretic mobility decreases with increasing the
electrolyte concentration, the Helmholtz-Smoluchowski formula in Equation 3.4 is used
to estimate the value of zeta potential. This is due to various concentration of solution
has changed the medium permittivity. Second condition is for zeta potential lower than
50 mV, the concentration of polarization is negligible and zeta potential calculated with
Equation 3.5 is accurate. In contrary, if the zeta potential is rather high, which is higher
than 50 mV, the Helmholtz-Smoluchowski formula is not applicable. However, this can
be solved with elaborating more models into the formula. For example, implementing
the numerical calculation of O’Brien and White [106] and formula derived by Dukhin
and Semenikhin [107]. Meanwhile, if κa is low or κa< 1, the Hückel–Onsager
Equation 3.6 is applied [2].
ηςεε
μ3
2 ore = (3.6)
For transition range between the low and high κa, Henry’ formula can be applied if the
zeta potential is assumed to be low (<50 mV) which the surface conductivity and
concentration polarization in this condition are negligible. For a nonconducting sphere,
Henry had derived the formula as in Equation 3.7.
( )ηκεςμ
32 af
e = (3.7)
where f(κa) is Henry’s function [108]. The f(κa) → 1, when κa → 0 and f(κa) → 3/2
when κa is near infinity. At intermediate values of κa, the value of f(κa) differs between
the different approaches. The value of f(κa) with 1.5 is used for electrophoretic
mobility that is commonly applied in aqueous medium and moderate electrolyte
concentration. For f(κa) with 1.5 is referred as the Smoluchowski approximation which
emphasizes for particles larger than about 0.2 microns dispersed in electrolytes that
contains more than 10-3 molar salt .
The unit of к is reciprocal length, which is 1/к and named Debye length. It is
denoted as the distance of counterions distribution around a charged particle in a
solution. The Debye length is considered as a measure of “thickness” of the electrical
double layer [109]. The parameter a, is referred to radius of the particle, and therefore
кa is considered a ratio of the particle radius to the Debye length, κ-1.
When κa>>1, the κ is defined as,
2/1
1
22
⎪⎪⎭
⎪⎪⎬
⎫
⎪⎪⎩
⎪⎪⎨
⎧
=∑=
kT
nzq
or
N
iii
εεκ (3.8)
with a the radius of the particle, q the elementary charge, z the charge number and ni i
number of ion concentration (the solution contains N ionic species), εr the relative
permittivity of the electrolyte solution, εo the electric permittivity of vacuum, k the
Boltzmann constant, and T the thermodynamics temperature.
Figure 3.2. Illustration of the Hückel and Smoluchowski limits of the Henry’s function where a, is the particle radius and к-1 is the Debye length or double layer thickness [70].
Hückel limit Smoluchowski limit
There is a limitation condition for Henry’s function which is represented in Figure
3.2 [110]. Henry’s function reduces to the Smoluchowski limit of 1.5. On the other hand,
if the particle radius is small compared to the Debye length, the value is reduces to the
Hückel limit of 1.0 from Henry’s function. The f(κa) term in the Henry equation is
known as the Henry function, and for a sphere in the presence of mobile ions is defined
as shown below.
For κa>1,
3322
3302
7529
23)(
aaaaf
κκκκ −+−= (3.9)
For κa<1,
∫−
⎥⎦
⎤⎢⎣
⎡−−+−−+=
aa dtt
eeaaaaaaafκ
α
κκκκκκκκ1645432
96)(
8)(
96)(
96)(
48)(5
16)(1)( (3.10)
Henry introduced two series expansions for the function of f, one for small κa and
another for large κa [111] . Ohshima [112] has provided an approximation analytical
expression which duplicates the Henry expansion almost exactly. Ohshima’s relation is
shown as below equation.
[ ] ⎥⎥⎦
⎤
⎢⎢⎣
⎡⎟⎟⎠
⎞⎜⎜⎝
⎛−+
++=−3
)exp(215.21
211)(
aaaf
κκκ (3.11)
The diffuse layer of the electric double layer enables the surface charge of a particle
to be estimated. Meanwhile, zeta potential is similar with this diffuse layer potential,
which can be measured with electrophoresis. Equations have been developed to support
the model and numerical calculation on the conversion of electrophoretic mobility to
zeta potential.
In this study, the increase of measurements repetitions and averaging of zeta
potential was carried out. Besides, polydispersity of the sizes and shapes of charged
particles for measurement should be minimized. The samples are all in spherical shape
except E.coli in this study. However, average results of the electrophoretic mobility of
E.coli were used to compare with the spherical charged particles and cells.
Besides the shape and sizes of the charged particles for electrophoretic mobility
and zeta potential measurement, the electrokinetic behaviour of charged particles is also
associated with solution conductivity and liquid transporting inside the charged particles
or cells. This increases the complication of surface charge study, which the cells might
swell depending on solutions. Thus, pH solution is very important to monitor the
surface charge stability of charged particles and cells.
The particle with partial penetrable characteristic may consider as a gel-like
corona, for instance liposome and bacteria. Comparison between the semi-solid particles
and solid particles can be studied to compare the changes of surface charge for the same
solution and measurement conditions. In addition to this, there is a model calculating
electric potential inside the gel layer, namely Donnan potential. It is represented by a
friction parameter incorporated in the Navier-Stokes equation and have been established
by Ohshima, which this theory is available for soft spheres ranges from solid particles
to penetrable particles [38].
The electrophoretic mobility and zeta potential in this study is measured with Laser
Doppler Velocimetry Electrophoresis system. The operational of the system in the
following chapter is further discussed in the next chapter together with the methodology
to prepare the samples.
CHAPTER 4
Methodology and Materials 4.0 Preparation of the Materials (Colloid Particles and Bacteria) In this chapter, the samples TiO2 and liposome were synthesized in colloid lab.
Meanwhile, the bacteria were culture and incubated for the experiments. The details of
the preparation were discussed below.
4.1 Solvent and Buffers Cyclohexane was purchased from Sigma-Aldrich which was used as an organic
solvent and surfactant of Triton X-45 (Sigma-Aldrich) was selected. Tetrapropyl
orthotitanate (Ti(OC H )3 7 4) from Fluka and sodium hydroxide (HmbG chemical) were
used. Buffer at pH 7 was prepared with Sodium Phosphate Monobasic Dihydrate
(H NaO P.2H2 4 2O) from Fluka, assay >>99.0% and Sodium Phosphate Dibasic Dihydrate
(HNa O P.2H2 4 2O) with a purity up to 98% was also purchased from Fluka. Capric Acid
(C10H20O2) (Fluka) and assay is more than 98%. Sodium hydroxide (HmbG) which is
insoluble in H TMO was used. Live and dead bacteria were cultured with CHROMagar2
O157 for E.coli and CHROMagarTM Staph aureus for S.aureus which were purchased
from LabChem Sdn.Bhd.
4.1.1 Preparation of TiO2
The calculation of molar ratio (wo=[H 2O]/[Surf]) which varied the sizes of the
synthesized TiO was determined. Nanosized TiO2 2 particles were prepared by
controlled the hydrolysis Tetrapropyl orthotitanate (TPOT) with 0.5 M of sodium
hydroxide in deionized water. Triton X-45 was mixed with cyclohexane as it is
dissolvable in organic solvent. The solution of microemulsion was kept stirring for ten
minutes and the size of the microemulsion was measured with zetasizer, nano-ZS. On
the other hand, the TPOT was mixed in cyclohexane and was added dropwise to the
microemulsion. The solution was stirred throughout the process for overnight. Ethanol
(95%) was added into the solution in the ratio of 3:2 (Ti(OH)4: ethanol) and stirred for
another 30 minutes to damage the emulsion and solubilize the surfactant. The solution
was dried at 60oC for 24 hours and followed by calcination at 550oC for 4 hours. Sizes
of the dried fine powder of the synthesized TiO were then examined. 2
4.1.2 Preparation of Fatty Acid Liposome
Liposome with final concentration of 20 mM at pH 7 was prepared with capric acid
(C10H20O2) and sodium hydroxide in 50 mM as stock. 50 mL of the solution was mixed
with approximately ten drops of hydrochloric acid with 1 M. A milky solution was
observed, thus liposome was formed. The pH level of the solution was checked and
fixed at pH 7 while the remaining volume was filled with deionized water up to 50 mL.
4.1.3 Preparation of Live E.coli and Live S.aureus
Two well characterized strains of Escherichia Coli (E.coli) O157 and
Staphylococcus aureus (S.aureus), ATCC 35983 were selected as model bacteria cells
for this surface charge study. These strains of bacteria were supplied from Microbiology
Lab at University Malaya. E.coli was tested with CHROMagarTM O157 and the colony
was in Mauve colour. CHROMagarTM Staph aureus for colony S.aureus appeared in
Rose colour. Both strains of bacteria were then cultured with plate agar to prepare for
the experiments. The bacteria were cultured in incubator for overnight at 37oC. This
temperature was the most suitable for both strains of bacteria to grow. The bacteria were
harvested after 24 hours and were pelleted by centrifugation for 15 min at 1500 g. The
growth medium for bacteria was agar plate, thus the bacteria was easy to clean off
before usage on experiment. The bacteria were suspended in buffer at pH 7.
4.1.4 Preparation of Dead E.coli and Dead S.aureus
The grown bacteria on the agar plate were incubated again at temperature of 55oC
for four hours. The bacteria were then harvested and centrifuged at the same rate as
mentioned above before ready for further test. Test was carried out before to confirm
the bacteria was dead with culturing the bacteria after being incubated for four hours at
55oC. There was no new growth of colony for 24 hours of incubation which confirmed
that the bacteria were dead. Temperature at 55oC was out of the suitable range for these
strains of bacteria to grow. However, new colony was found if the heating hours were
less than four hours. Consequently, the bacteria was dead without denature in this time
duration with heating at 55oC.
4.2 Introduction to Laser Doppler Velocimetry Electrophoresis System In this study, electrophoretic mobility of the particles was determined by using
Zetasizer series nano-ZS from Malvern Instrument. The electrophoretic mobility
measurement is directly obtained with the conversion to zeta potential from theoretical
considerations. The conventional system of micro-electrophoresis is a cell with
electrodes at the end to which a voltage is supplied. When particle moves towards the
electrode of the opposite charge, the velocity is measured and the mobility expressed in
unit field strength. The cell used in this instrument is in folded capillary cell with
electrode at both ends which is shown in Figure 4.1. The technique for mobility
measurement for Malvern series is known as Laser Doppler Velocimetry
Electrophoresis System.
Figure 4.1. The folded capillary cell for electrophoretic mobility measurement which is designed by Malvern’s Zetasizer Nano Series [2].
Laser-Doppler Velocimetry (LDV) Electrophoresis system is a method for
measuring fluid velocities by detecting the Doppler frequency shift of laser light that
has been scattered by small particles moving in the flow. The Doppler frequency shift is
the difference between that of the incident laser beams and the scattered light
frequencies. This technique was first used in water flows by Yeh & Cummins [113] and
has since seen significant improvements, in the signal processing, in particular. A
review of the advances in LDV can be read in Tropea [114]. The technique remains a
complicated procedure and requires some understanding of the basic principles. A more
complete review of the theory is described by Durst et al. [115].
4.2.1 Overview on Electrophoretic Light Scattering
The light scatters at an angle of 17o and is combined with the reference beam. This
produces a fluctuating intensity signal where the rate of fluctuation is proportional to the
speed of the particles. A digital processor is used to extract the characteristic
frequencies in the scattered light, which is shown in Figure 4.2.
Figure 4.2. The occurring of the light scatters at angle of 17o and produces the intensity of the scattered light.
In electrophoretic light scattering, the velocity of a charged particle under the
influence of an applied electric field is measured by monitoring the frequency shift (Δf)
of the light scattered from the particle. The light scatters from the stationary particle will
have the same frequency and wavelength as the incident light, whereas the light scatters
from particles in motion will be Doppler shifted. In this system, the electrophoretic
mobility of the particle is calculated from the Doppler frequency shift which is
demonstrated in Equation 4.0,
λ
θ
μ⎟⎠⎞
⎜⎝⎛
=Δ 2sin
2 ef (4.0)
where μe is denoted as the electrophoretic mobility, θ is the scattering angle and λ is the
wavelength of the incident light.
The light beam or the incident ray consists of laser with 633 nm with frequency, 4.7
× 1014 Hz. The intensity that is produced is a quantum of light which equivalent to the
square of the wave amplitude. The scattering intensity is monitored by using an
Avalanche Photo Diode (APD) detector. The response time for an APD is in the order
of 0.1 μs which corresponds to 1 × 107 Hz. This has provided seven orders of magnitude
different between the light and detector response frequencies, thus an APD cannot be
used to directly measure the frequency of the scattered light. However, this can be
solved by using a method name heterodyning; meanwhile the APD can be used to
indirectly measure the frequency shift of the scattered light.
In this heterodyning method, a fraction of the incident light is mixed with the
scattered light to generate a “beat” pattern, the frequency of which can be easily
monitored with a time dependent intensity trace from an APD. This method is shown in
Figure 4.3, which demonstrate two waves of slightly different frequencies (Base wave
and Frequency Shifted) along with the summation of the two. The summation produces
wave which pulses or beats at a lower frequency compared to that of the other two
components.
A refinement of the system involves modulating one of the laser beams with an
oscillating mirror which gives an unequivocal measure of the sign of the zeta potential.
Another advantage of this optical modulator is that low or zero mobility particles give
an equally good signal, which the measurement is as accurate as for particle with a high
mobility. This technique ensures an accurate result in a matter of seconds with possibly
millions of particle can be observed [2].
Figure 4.3. Two waves of slightly different frequencies (Base wave and Frequency Shifted) along with the summation of the two.
Base wave Frequency shifted Summation
4.2.2 Advantages of LDV to overcome Electroosmosis Effect
Electroosmosis is phenomenon about the movement of a liquid relative to a
stationary charged surface under the influence of an electric field. Most of the
microelectrophoresis system had reported the electroosmotic flow occurs for those
particles move near the wall of the capillary cells when electric field is applied.
However, in a closed system, the flow along the walls must be compensated by a
reverse flow down the center of the capillary. There is a point in the cell at which the
electroosmotic flow is zero where the two fluid flows cancel. Thus, any measurement
performs at this point is the genuine electrophoretic velocity. This point is called the
stationary layer and is the spot where the two laser beams cross. Consequently, the zeta
potential measured is therefore free of electroosmotic errors which can be seen from
Figure 4.4 [116].
Figure 4.4. Stationary layer where two laser beams cross is free from electroosmotic effect.
Laser-Doppler Velocimetry (LDV) Electrophoresis system is a method for
measuring fluid velocities by detecting the Doppler frequency shift of laser light that
has been scattered by small particles moving in the flow. The Doppler frequency shift is
the difference between that of the incident laser beams and the scattered light
frequencies. This technique was first used in water flows by Yeh & Cummins [113] and
has since seen significant improvements, in the signal processing, in particular. A
review of the advances in LDV can be read in Tropea [114]. The technique remains a
complicated procedure and requires some understanding of the basic principles. A more
complete review of the theory is described by Durst et al. [115].
4.2.3 Introduction on M3-PALS Technique
Zetasizer Nano series from Malvern instrument [116] introduces the latest
technology which combining the technique of Laser Dopper Velocimetry and Phase
Analysis Light Scattering, abbreviated as M3-PALS technique [1]. The M3 technique
consists of both Slow Field Reversal (SFR) and Fast Field Reversal (FFR) measurement;
hence it is a “Mixed Mode Measurement”. The advantage of M3 is the above mentioned
measurement is in sequence for each measurement. It is no longer necessary for the
operator to select any system parameters for the measurement. With a reduction in the
number of measurement variables, both the measurement repeatability and accuracy are
improved. Furthermore, the alignment of the system to focus at the location of the
stationary layer is no longer a problem.
The Slow Field Reversal (SFR) is applied to reduce the polarization of the
electrodes which is inevitable in a conductive solution. The frequency for this field is 1
Hz. On the other hand, there is another field applied to the measurement, namely Fast
Field Reversal (FFR). This field is reversed more rapidly compare to Slow Field
Reversal until it is possible to show that the particles reach terminal velocity in fluid
flow due to insignificant electroosmotic. The mobility measured during this period is
purely on the electrophoresis of the particles and is not affected by electroosmosis. The
frequency for FFR is 30 Hz.
The sequence of these M3-PALS is due to the first measurement is performed at the
cell centre and this gives an accurate determination of the mean. This is followed by a
Slow Field Reversal measurement, which gives better resolution but the mobility values
are shifted by the effect of electroosmotic. The mean zeta potentials calculated from the
FFR and SFR measurements are subtracted to determine the electroosmotic flow and
this value is then used to normalize the Slow Field Reversal distribution. The value for
electroosmotic is used to calculate the zeta potential of the cell wall.
Phase Analysis Light Scattering uses phase shift. This phase is preserved in the
light scattered by moving particles but is shifted in phase in proportion to their velocity
and this phase shift if measured by comparing the phase of the light scattering by the
particles with the phase of a reference beam. A beam splitter is used to extract a small
proportion of the original laser beam to use as the reference beam. The detection of a
phase change is sensitive to changes in mobility [116].
4.2.4 Operation of Zetasizer for Mobility Measurement
The Zetasizer Nano Series for zeta potential or electrophoretic mobility
measurement consists of six main components. The arrangement of the system can be
depicted in Figure 4.5. This is the detail description of the system. But the instrument
was compacted in a plastic box as can be seen in Figure 4.6.
The first part was laser source as light source to illuminate the particle within the
sample. This light source was splited to provide an incident and reference beam. At the
same time, the reference beam was modulated to give the Doppler Effect necessary. The
laser beam was then passed through the sample cell which was the second component. It
was scattered at an angle of 17o and was detected. Any particles moved through the
measurement volume would cause the intensity of light detected to fluctuate with a
frequency proportional to the particle speed when an electric field was applied.
Component three was the detector that sent the information to a digital signal
processor, which was the fourth component. The next component was the computer,
where the Zetasizer Nano software produced a frequency spectrum from which the
electrophoretic mobility and zeta potential were calculated. Component sixth was an
attenuator to reduce the intensity of the laser light which to avoid overloading the
detector. Lastly, the scattering beam path was installed which was called compensation
optics to correct for any differences in the cell wall thickness and dispersant refraction.
It was worked as the alignment system. For electrophoretic mobility and zeta potential
measurement, folded capillary cell where used [116].
Figure 4.5. The arrangement of the components for zeta potential measurement with Zetasizer Nano series instrument [1].
Figure 4.6. The components show in Figure 4.5 except computer was compacted in this plastic box with the mentioned size [1].
CHAPTER 5
Effects of Different chemical environments on the Electrophoretic Mobility
5.0 The magnitude of the electrophoretic mobility of bacteria and colloidal particles in different chemical environment and salt effects
The magnitude of the electrophoretic mobility of charged particles and cells is
greatly affected by the suspension with varied pH and salt concentration. In this chapter,
the emphasis is on measuring the change in the electrophoretic mobility of bacteria and
colloidal particles after numbers of centrifugation, in distilled water and in different
chemical conditions.
The electrophoretic mobility of all samples suspended in distilled water was
measured. As distilled water is a hypotonic medium to bacteria, hence, prolong
immersion in distilled water might lyse bacteria due to osmosis in which water enters
cells. Thereby, bacteria were suspended in distilled water for 10 minutes before
measurements can be carried out. Another factor that could have affected the surface
charge of bacteria was numbers of washing. This is discussed later in this chapter.
Various ionic and pH strengths of a suspending medium have been reported to
affect the surface charge of charged particles and biological cells [1, 2, 3]. The surface
cgarge is also highly depended on ionic strengths of a suspension medium. In this study,
the effects of different pH condition from pH 2-10, concentrations of sodium chloride
(NaCl) and ammonium chloride (NH4Cl) ranging from 0.01 to 0.08 M were investigated.
In addition, sizes of bacteria and colloidal particles in this study are shown. Comparison
of the colloidal particle, TiO was used as a reference sample in this chapter. 2
5.1 Size distribution of E.coli and S.aureus The transmission electron micrograph of E.coli and S.aureus were obtained by
using transmission electron microscope. The micrograph of E.coli with the size
measurement is depicted in Figure 5.1 (a). One unit of E.coli displays in the figure is a
rod-shape bacterium, with width of 686 nm and length of 1560 nm. The size ratio of
length to width is about 2. On the other hand, S.aureus is grape-like bacteria which
show an average diameter of 821 nm. This is shown in Figure 5.1 (b).
The bacteria in the figures were initially washed three times and a series of
treatment before trimmed to view the micrograph. Consequently, flagella on E.coli were
washed off during the centrifugation process.
Figure 5.1 (a). The micrograph of E.coli O157:H7.
100nm
Figure 5.1 (b). The micrograph of S.aureus.
100nm
5.1.1 Size distribution of TiO2 and liposome 5.1.1 Size distribution of TiO2 and liposome The particle size distributions for TiO2 and liposome are depicted in Figure 5.2 with
an average size of 255 nm and 164 nm respectively. Polydispersity of TiO
0
10
20
30
40
0.1 1 10 100 1000
Inte
nsity
(%)
Size (d.nm)
TiO2, 255 nm
Liposome, 164 nm
Figure 5.2. Diameter sizes of the liposome and TiO2
2 particle was
lesser than liposome, indicating particles size of TiO2 were more consistent. A
transmission electron micrograph of TiO2 and polarizing microscope micrograph of
liposome are shown in Figure 5.3 (a) and (b).
The particle size distributions for TiO2 and liposome are depicted in Figure 5.2 with
an average size of 255 nm and 164 nm respectively. Polydispersity of TiO2 particle was
lesser than liposome, indicating particles size of TiO2 were more consistent. A
transmission electron micrograph of TiO 2 and polarizing microscope micrograph of
liposome are shown in Figure 5.3 (a) and (b).
2 µm 2 µm
Figure 5.3 (a) Micrograph of TiO2 (b) Micrograph of caprate-capric acid liposome
5.2 Effects of number of washes on surface charge of live E.coli and S.aureus
It have been reported that a high speed centrifugation of 10,000 g reduced the
electrophoretic mobility of bacteria. However, this depends on different centrifugation
protocols and strain of bacteria [117]. Simple centrifugation is meant to remove
chemicals from the culture medium before running any test [118, 119].
The change of surface charge after numbers of washing of live E.coli and S.aureus
were measured and are depicted in Table 5.1. The bacteria which were suspended in
phosphate buffer of pH 7 solution and centrifuged at a speed of 6000 g for 30 min
showed increasing in electrophoretic mobility. This might due to the ions in the solution
has not been able to adhere firmly on the surface of charged particles especially the ion
distribution at diffuse layer while the charged particles move with fast rotation.
Besides, another factor that increasing electrophoretic mobility as number of wash
increased might be caused by the settlement of heavier bacteria to the bottom of the tube.
This leaves the lighter and smaller bacteria suspends in the solution and latter were
extracted for measurement. Hence, smaller-sized bacteria can migrate faster as electric
field is supplied and resulted in an increase in the magnitude of the electrophoretic
mobility.
EPM of E.coli
EPM of S.aureus Numbers of
washing m2 V-1 s-1
Standard Deviation m2 V-1 s-1
Standard Deviation
Unwash -1.03 0.43 -2.23 0.41 Wash 1 -1.39 0.24 -2.87 0.66 Wash 2 -1.70 0.71 -3.35 0.53 Wash 3 -1.81 0.33 -3.95 0.42
Table 5.1. The magnitude of the electrophoretic mobility of live E.coli and S.aureus after number of wash.
The result shows that the negativity of the magnitude of the electrophoretic
mobility for live bacteria (E.coli and S.aureus) increased with the number of washes at
6000 g revolutions per minute for 30 minutes. A 6000 g revolution per minute for the
centrifugation was determined to prevent the cell breaking of bacteria. In addition,
Table 5.1 shows that the negativity of the magnitude of the electrophoretic mobility of
S.aureus was greater overall than E.coli. This is because the gram-positive S.aureus has
a thicker cell wall to prevent excessive loss of ions from the cell when centrifugation is
carried out.
5.3 The magnitude of the electrophoretic mobility of bacteria and TiO2 in distilled water
The changes of surface charge bacteria in distilled water were investigated. Cell
membrane is inferred to lyse after suspended a long time in distilled water, namely
hypotonic solution. Thus, bacteria were suspended in distilled water for 10 minutes for
this measurement. Meanwhile, TiO2 as solid colloidal particles were suspended in
distilled water to measure the magnitude of the magnitude of the electrophoretic
mobility and it was used as a reference sample to compare with biological cells.
Table 5.2 shows the magnitude of the electrophoretic mobility of TiO2, live and
dead bacteria suspended in distilled water for 10 minutes before measurement. The
EPM
Samples m2 V-1 s-1
Standard Deviation
TiO2 -3.02 0.32 Live E.coli -0.76 0.56 Dead E.coli -0.81 0.46
Live S.aureus -2.24 0.38 Dead S.aureus -2.71 0.43
Table 5.2 The magnitude of the electrophoretic mobility of TiO2, live, dead E.coli and S.aureus suspended in distilled water.
results indicated that live and dead E.coli exhibited the lowest electrophoretic mobility
than TiO2 and S.aureus. There are two factors might be the causes of low
electrophoretic mobility for E.coli. First, size of E.coli is the largest among the samples.
The larger size of E.coli led to a lower electrophoretic mobility due to the slower
response to applied field compared with cells of smaller size. Secondly, the size of
S.aureus is much smaller than E.coli and has a five to ten times faster diffusion rate than
E.coli for uncharged small particles to penetrate through the cell. However, distilled
water constitutes of charged molecules would attract to the negatively charged cells
instead of penetrating through the cell. Hence, smaller size of S.aureus and with a
thicker peptidoglycan layer than E.coli adsorbs more positive ions towards the surface
cells. The positive ions are contributed by hydrogen ion of water molecules, leaving
oxygen ions bonded at the water molecules to face outwards. This increases the net
negative charge of S.aureus and, thus higher the magnitude of the electrophoretic
mobility was obtained.
On the other hand, TiO2 obtained the highest electrophoretic mobility compare
to other samples. This is because of the smallest size of TiO2 is able to response faster
whenever electric field is applied. Besides, TiO2 is the only solid colloidal particle that
not penetrated by water molecules. Hence, the net negative surface charge of the
particles is not much affected in distilled water suspension. Furthermore, the adsorption
of positive side of water molecules to the surface of TiO2 increases electrophoretic
mobility.
The magnitude of the electrophoretic mobility of S.aureus in distilled water did
not differ much from TiO . This indicated that the negative surface charges live and 2
dead S.aureus were not much affected in distilled water suspension for duration of 10
minutes.
5.4 The electrophoretic mobility of biological cells and colloidal particles in different pH
Stability of Surface charge of biological cells and charged particles depends on
pH solution. It has been reported that the negative electrophoretic mobility of charged
particles increases with increasing pH value, that is caused by the higher concentration
of hydroxide ions in suspension at higher pH. The negatively charged hydroxyl ions
reduce the neutralization effects on the negatively charged particles or cells, which leads
to the increase in magnitude of the electrophoretic mobility. In contrast, cells or charged
particles in pH of less than 7 are concentrated with hydrogen ions (H+) and this increase
the screen effect on the negatively charged particles. Hence, any further addition of acid
to the suspension can cause building-up positive surface charge of particles and cells.
Isoelectric point is a point, where zero zeta potential is obtained. It has been
reported that the isoelectric point for most of gram-positive and -negative bacteria is at
pH 1-4 [120]. The Malvern’s manual of zetasizer also reports similarly the isoelectric
point of zeta potential at pH 5.5 [68]. This is very essential point to determine the
stability of charged particles due to the least stable point for charged particle with a zero
zeta potential. As a consequent, unstable surface charge particles cause agglomeration
as the repulsion force between charged particles reduces.
In this study, phosphate pH buffer solutions ranging from pH 2 to 10 were used to
suspend samples. Viability of live bacteria at this pH range was studied by preparing a
population of bacteria and suspended in phosphate buffer at different pH for an hour
before the bacteria was cultured on agar plates. After an overnight of incubation, new
growth of bacteria was found except for bacteria which were suspended in phosphate
buffer of pH 2 to 4. Since the magnitude of the electrophoretic mobility of live and dead
bacteria is compared, hence this test is important to ensure that the live bacteria survive
in the pH suspension.
5.4.1 Comparison of the magnitude of the electrophoretic mobility of live and
dead E.coli (TiO as reference) 2 Comparison of the magnitude of the electrophoretic mobility of live and dead E.coli,
together with TiO2 as reference sample is shown in Figure 5.1. Live and dead E.coli
demonstrated an increase in the magnitude of the electrophoretic mobility at different
pH. The result shows that the magnitude of the electrophoretic mobility of E.coli was
more positive at lower pH. This is because the concentration of hydrogen ions was
sufficiently high at lower pH to lower the net negative charge on the surface.
Meanwhile, isoelectric points of live and dead E.coli were obtained at pH 2.
By comparing the magnitude of the electrophoretic mobility of solid colloidal
particle, TiO2 with E.coli, a higher magnitude of electrophoretic mobility was obtained
for TiO2 than both live and dead E.coli. However, the magnitude of the electrophoretic
mobility of TiO2 decreases at lower pH, namely from pH 2 to 4, but increased
drastically after pH 4. This showed that the increase in concentration of hydroxide ions
at higher pH reduced the neutralization effects on negatively charged surfaces of
charged particles. This increases the net negative charge on surfaces and leads to a
higher magnitude of electrophoretic mobility for the charged particles. However, the
result shows a decline in magnitude of electrophoretic mobility from pH 8 to 10. This is
due to the greater concentration of hydroxyl ions at higher pH increases the conductivity
of ions in suspension, which then surpassed the conductivity of the charged particles
when electric field is applied. This reduced the movement of the charged particles and
lowers its magnitude of electrophoretic mobility at higher pH.
Figure 5.4. Comparison of the magnitude of the electrophoretic mobility live and dead E.coli (TiO2 as reference sample) at various pH (Polynomial least square analysis is
The
elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10-8
)
pH
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.00
0.50
1.000 2 4 6 8 10 12
TiO2Live E.coliDead E.ColiPoly. (TiO2)Poly. (Dead E.Coli)Poly. (Live E.coli)
The magnitude of the electrophoretic mobility of live E.coli was observed to be
erratic at various pH, which depicted in Figure 5.4. The increase in magnitude of the
electrophoretic mobility of dead E.coli at various pH was slight but displayd a rather
stable increased in magnitude from pH 2 to 10. On the other hand, the magnitude of the
electrophoretic mobility of live E.coli increased from ~ (-0.5 ± 0.1) × 10-8 m2 V-1 s-1 to ~
(-1.0 ± 0.1) × 10-8 m2 V-1 -1s from pH 2 to 4, but decreased to ~( -0.5 ± 0.1) × 10-8 m2 V-1
-1s at pH 5 and 6. This was followed by a sudden increase in magnitude at pH 7 and a
decline in magnitude from pH 8 to 10. This showed that surface charge of live E.coli
was stable in pH 7. On the other hand, the total net change in electrophoretic mobility
from pH 2 to 10 for dead and live E.coli were ~ (1.0 ± 0.1) × 10-8 m2 V-1 s-1 and ~ (1.3 ±
0.1) × 10-8 m2 -1 -1V s respectively. The slight greater change of net negative surface
charge for live E.coli was shown.
This study demonstrated the difference in changes of electrophoretic mobility
for live, dead E.coli and TiO2. This reflected unique surface charge properties of
biological cellular activity and surface structure [120]. The difference between live and
dead E.coli may be attributed to biochemical mechanisms and active metabolic activity
in live cells. The erratic magnitude of the electrophoretic mobility of live E.coli
throughout the pH range indicated the resistance of change on surface charge for live
cell, which unlike dead E.coli had lost the metabolic activity to recharge the cells after
the screen effect in different suspension. Consequently, dead E.coli gave a more
systematic increase in magnitude of electrophoretic mobility in this pH range.
5.4.2 Comparison of the magnitude of the electrophoretic mobility of live and
dead S.aureus (TiO as reference) 2 The magnitude of the electrophoretic mobility of live and dead S.aureus at a range
of pH solution was compared. The magnitude of the electrophoretic mobility of TiO2
was used as a reference sample to differentiate the response of biological cells and
colloidal particle in different pH. Figure 5.5 shows the magnitude of the
electrophoretic mobility of live and dead S.aureus intersecting at pH 4. This is due to
live S.aureus was dead after being suspended in phosphate buffer at pH 2 to 4 after one
hour. This causes the result of the magnitude of the electrophoretic mobility of live and
dead S.aureus was hard to compare at pH 2 to 4.
TiO2 recorded a more positive magnitude of the electrophoretic mobility at pH 2
to 4 than S.aureus. Meanwhile, isoelectric point of TiO2 was observed at pH 2. This is
because the negative charges on TiO2 were neutralized by hydrogen ions in the acidic
environment. However, live and dead S.aureus obtained a higher magnitude of
electrophoretic mobility than TiO2. This indicated that the screens effect of TiO2 at
lower pH is greater than S.aureus.
The magnitude electrophoretic mobility of live S.aureus was more negative than
dead S.aureus from pH 4 to 10. Since live S.aureus is metabolically active, thereby the
negative surface charges of live cells are more stable than dead S.aureus. In addition,
the thicker cell wall of live S.aureus can prevent surrounding particles from penetrating
through the cells, this has lessen the change of net negative charge on it compare to
dead cells. In contrary, dead S.aureus is metabolically inactive, which is not able to
rejuvenate the negative surface charges of cells, thus leading to a more positive
electrophoretic mobility than live S.aureus.
The effects of various pH on the magnitude of the electrophoretic mobility of
samples was obvious. The results showed that the magnitude of the electrophoretic
mobility of TiO2, live and dead S.aureus increased as acidicity of the surrounding
environment decreased. However, the results also indicated a slight reduction in
magnitude of electrophoretic mobility at highly alkaline environments, namely pH 9 to
10. This is due to the higher concentration of hydroxyl ions at pH 9 and 10 surpasses the
conductivity of charged particles, subsequently lower the magnitude of the
electrophoretic mobility. The negatively-charged ions responded faster to an electric
field compared to the negatively-charged cells or particles which were relatively more
massive.
Figure 5.5 . Comparison of the magnitude of the electrophoretic mobility live and dead S.aureus (TiO2 as reference sample) at various pH (Polynomial least square analysis is shown).
The
elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
pH
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.00
0.50
1.000 1 2 3 4 5 6 7 8 9 10 11 12
TiO2Live S.aureusDead S.aureusPoly. (Dead S.aureus)Poly. (Live S.aureus)Poly. (TiO2)
The total change in electrophoretic mobility against pH for live S.aureus was
(1.5 ± 0.1) × 10-8 m2 V-1 -1s . Meanwhile, the total net change in electrophoretic mobility
of dead S.aureus was lower than live S.aureus, namely, (0.8 ± 0.1) × 10-8 m2 -1 -1V s .
These results indicated that the effects of pH were greater on live S.aureus than dead
S.aureus.
5.4.3 Comparison of the magnitude of the electrophoretic mobility of live E.coli and S.aureus at different pH
In the previous topic, comparison was done on the same strains of bacteria for
live and dead population. Meanwhile, comparison of different strain at range of pH
showed that the magnitude of the electrophoretic mobility of E.coli was more erratic as
pH increased than S.aureus. This is due to live E.coli constitutes of a thinner
peptidoglycan and more proteins integrity of the cell increased the neutralization rate of
the negative surface charge. Meanwhile, S.aureus responded proportionally to the
increase of pH. This might due to thicker cell wall and smaller sizes of S.aureus
compared to E.coli adsorbs more hydroxyl ions on the surface, which increase the net
negative surface charge gradually as pH increases.
5.4.4 Conclusion on the effects of pH on surface charge of live and dead bacteria (TiO as reference sample) 2
A buffer solution of pH 2 to pH 6 is acidic and defined as a compound that
donates hydrogen ions (H+). On the other hand, a buffer solution of pH 8 to 10 is
alkaline and has higher concentration of hydroxide ions (OH-) in the solution. In acidic
buffer solution, negatively-charged surface charge particles adsorb positive ions, thus
neutralizing their surface charges and hence, reducing their response towards the
electric field. As a result, more positive values of electrophoretic mobility would be
obtained. This was shown by the result of electrophoretic mobility of live, dead E.coli
and S.aureus, together with TiO2 as a reference sample.
The number of ions on the surface of live cells is manipulated by ion-pumping
systems. Thereby, number of positive ions from the solution that adsorbed onto the
surface charge of cells would be recovered in return. This was shown from the erratic
electrophoretic mobility of live E.coli at different pH in response to electric field. The
highest electrophoretic mobility of live E.coli was at pH 7, neutral pH. This indicated
that live E.coli experienced less stressed in this solution. The net negative charge of
E.coli remained in this solution. Dead E.coli showed a more sequence increase in this
pH range as the dead cell were metabolically inactive and would not rejuvenate the net
negative charge on the surface.
It was also observed that live and dead S.aureus possessed higher electrophoretic
mobility at pH 2 to 4 compared to TiO . This indicated that the screens effect on TiO2 2 at
lower pH was obvious. On the other hand, the magnitude of the electrophoretic mobility
of dead S.aureus did not changed much from pH 2 to pH 7 due to increase in
permeability of dead cell membrane to let ions moved in and out. Meanwhile, dead cells
increased more drastic in electrophoretic mobility from pH 8 to pH 10 due to the
increase of negative ions in the solution which, in turn, thickened the diffuse double
layer and thereby obtaining greater the magnitude of the electrophoretic mobility. In
comparison, live S.aureus increased in electrophoretic mobility across the whole range
of pH. This is due to their thicker and rigid cell wall of gram-positive bacteria enabled
them to give a better response to the change of ions at different pH. As a consequent,
the negative charge on the surface layer reduced the screen effects at lower pH and the
diffuse double layer of the cells would have been more compressed at higher pH, thus
increasing the magnitude of the electrophoretic mobility of cells.
The results in Figure 5.4 and 5.5 showed that live and dead E.coli possessed
lesser net negative surface charge compared to live and dead S.aureus. It is due to the
smaller size of the S.aureus compare to E.coli is easier to migrate towards the electode
as electric field is applied. The main reason might due to the different thickness of the
peptidoglycan layer of the cell wall and protein composition. The thicker cell wall can
prevent cellular ions from being lost to the outer layer and minimized uncharged
particles influx into the cells. Hence, most of the oppositely charge ions would be
attracted towards the surface charge of the cells. This increases the net negative charge
of S.aureus than E.coli. Like the surface charge properties of TiO2, the amount of
negative charges on the surface of S.aureus increased the magnitude of the
electrophoretic mobility during a change in pH. However, solid particles such as TiO2
conducted no exchange of ions in and out of them, thus obtaining the highest
electrophoretic mobility compared to E.coli and S.aureus.
5.5 Effects of different salt on the magnitude of the electrophoretic mobility of live and dead E.coli and S.aureus (TiO2 as reference sample).
Types of electrolytes and electrolytes concentration directly influence the
magnitude of the electrophoretic mobility of colloidal particle and cells suspension.
Effects of different salt concentration and salt type were investigated. Two types of salts,
sodium chloride and ammonium chloride that later dissolved in phosphate buffer pH 7
solution were investigated and electrophoretic mobility were measured. The salts
dissolved in the buffer solution will completely dissociated into ions in water and
formed strong electrolyte. Studies have reported that the types of electrolyte will
influence the conductivity of solutions [121, 98, 122].
Electrolyte solutions conduct electric current by migration of ions to electrodes
when an electric field is applied. This obeys Ohm’s law (5.1), where V is applied
voltage (V) , I denotes current (A) and R is resistance (Ω).
IRV = (5.1)
-1The conductance, L (S, Siemens, Ω ) is then defined as the reciprocal of resistance.
RL 1= (5.2)
Conductance of a liquid sample decreases when distance between the electrodes
increases but increases as the effective area of the electrodes increases. This is shown in
Equation 5.3.
lAkL = (5.3)
where k is the conductivity (S m-1), A is the cross-sectional area of electrodes ( the
effective area that electrons conduct in liquid), and l is the distance between the
electrodes. Molar conductivity is defined as in Equation 5.4.
ck
m =Λ (5.4)
where c is the molar concentration of the added salt of electrolyte. The molar
conductivity is independent of concentration if k is proportional to concentration of
electrolyte. However, in practice, molar concentration always varies with electrolyte
concentration [122]. Friedrich Kohlrausch discovered the empirical relationship
between the molar concentration of a strong electrolyte and the molar conductivity at
low concentrations as shown in Equation 5.5.
cKmm −Λ=Λ 0 (5.5)
where K is a non-negative constant depending on the electrolyte and Λ0m is the limiting
molar conductivity. Consequently, the conductivity of electrolyte can be determined.
The electrophoretic mobility, µ is given in the formula as shown in Equation 5.6.
26 rZπη
μ = (5.6)
A study has reported that there are few variables that usually determine electrophoretic
mobility as demonstrated in Equation 5.7 [32], where Z is valence of the charge, η is
viscosity, I is the ionic strength of electrolyte and r is the radius of the charged particle.
IrZ
72 1030.31.
6 ×=
πημ (5.7)
2/1−I Equation 5.7 indicates that a plot of µ versus will be linear for any
migrating analyst, provided that temperature and ionization remain constant and, hence,
the Debye-Hückel limiting law is valid.
5.5.1 Effects of ammonium chloride (NH4Cl) on the magnitude of the electrophoretic mobility of TiO and bacteria 2
+Studies have shown that many substances containing ammonium ions (NH4 )
were used to investigate the viability effects on bacteria since nitrogenous substances
are normally preferred by most of micro-organisms [123] because these substances
helps to enhance the growth rate [106] of microorganisms. Meanwhile,
energy-dependent transport systems for ammonium ions (NH +4 ) have been widely
discussed. It also been reported that the effects of dioctadecyldimethyl-ammonium
bromide (DODAB) on the magnitude of the electrophoretic mobility of E.coli and
S.aureus, indicating that the negatively-charged cells were perfectly viable whereas the
positively charged cells did not survive in this chemical [106]. Since the ammonium
ions susceptibility on bacteria has been widely investigated, hence, ammonium chloride
(NH4Cl) has been selected to dissolve in pH 7 solutions to study the effect of this salt on
electrophoretic mobility of cells and colloidal particles. NH4Cl ranging from 0.01 M to
0.08 M was prepared, which NH4Cl in aqueous medium is slightly acidic. The ionic
dissociation of NH Cl is shown below. 4
+
NH4Cl ↔ NH + Cl- 4
The increase in ionic strength NH4Cl increased conductivity of solutions with
suspended materials at an applied electrical field of 20 V.
0
2
4
6
8
10
12
14
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Λ
, C
ondu
ctiv
ity
,
TiO2 Live S.aureus
Dead S.aureusLive E.coli
Dead E.coli
Figure 5.6 shows the plot of conductivity versus concentration of ammonium
chloride solution of TiO2, live and dead E.coli and S.aureus. Charged particles and cells
demonstrated an increase in conductivity as concentration of ammonium chloride
increased. Besides, conductivity of all samples showed the sequential increases with
concentrations of NH4Cl, indicating that electrolyte conductivity depends on the salt
concentration, regardless of the types of suspended materials. The weak salt was
partially dissolved in water, reducing the conductivity of electrolyte. In contrast, strong
c ,Concentration
Figure 5.6. Conductivity, Λ of TiO2, live and dead E.coli and S.aureus in different concentrations, c of NH4Cl.
electrolyte had complete ionic dissociation in water, forming a strong current when an
electric field was applied.
5.5.2 Effects of NH4Cl on the magnitude of the electrophoretic mobility live and
dead E.coli (TiO as reference sample) 2
Comparison of electrophoretic mobility of live and dead E.coli with TiO2 as a
reference sample in different ionic strengths of NH4Cl was carried out. The magnitude
of the electrophoretic mobility, µ, of TiO 2/1−I, live and dead E.coli versus 2 was
plotted and shown in Figure 5.7. The result showed that electrophoretic mobility, µ was
proportional to 2/1−I , corroborating equation 5.7 and yielding an expected best-fit line.
The results indicated the proportional increase in magnitude of electrophoretic mobility
at lower ionic strength but decreased in magnitudes at higher ionic strength of NH Cl. 4
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.000.00 2.00 4.00 6.00 8.00 10.00 12.00
TiO2Live E.coliDead E.coliPoly. (TiO2)Poly. (Live E.coli)Poly. (Dead E.coli)
1/ I
The
elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10-8
)
Figure 5.7. The magnitude of the electrophoretic mobility of live and dead E.coli (TiO2 as reference sample) versus the reciprocal of the square root of the ionic strength of NH4Cl. The dots with error bars are experimental points, the best-fitted continuous and dashed lines were plotted with second order of polynomial least square analysis.
The magnitude of the electrophoretic mobility of live E.coli increased as ionic
strength NH Cl decreased. The increase of ionic strengths NH4 4Cl increased the amount
of free ions of solution, which led to an increase of current of the electrolyte. As a result,
the magnitude of the electrophoretic mobility of samples decreased, as the share of the
current carried by samples declined as free ions in the electrolyte increased. In addition,
the increase of free ions in solution causes greater heat production. In contrast, low
buffer ionic strength reduces the overall current in electrolyte and results in less heat
production [124]. Hence, the magnitude of the electrophoretic mobility increases as the
ionic strength decreases.
The negative surface charge of live E.coli in ionic strengths of NH4Cl solution
was found to be greater than dead E.coli. This might due to protein integrity for dead
E.coli starts to decompose and also dislodge the protein from the cells. Hence, the net
negative charge on dead cell is lessening, led to a lower electrophoretic mobility
compare to live E.coli. On the other hand, measurement showed that the magnitude of
electrophoretic motilities of live and dead E.coli in NH4Cl solutions were lower
compared to TiO2, solid colloidal suspension as depicted in Figure 5.7. This may be
attributed to solid sphere of TiO2 is impenetrable to ions, which enable to remain the net
negative charge on surface and adsorb oppositely charges as counterions. In addition,
smaller size of TiO is able to move faster than E.coli when electric field is applied. 2
Live E.coli showed erratic result of the magnitude of the electrophoretic
mobility at different ionic strengths of NH4Cl, and this might have been due to minor
impurities in the solution. Meanwhile, dead E.coli, which had with a thinner cell wall
and was metabolically inactivated, hastened the screening effects of ions on the surface
charge of cells. This explained the greater positive the magnitude of the electrophoretic
mobility of dead E.coli compared to live E.coli.
5.5.3 Effects of NH4Cl on the magnitude of the electrophoretic mobility of live
and dead S.aureus (TiO2 as reference sample)
The magnitude of the electrophoretic mobility of gram positive S.aureus
decreased as ionic strength of NH4Cl increased, which is depicted in Figure 5.8. This
result was similar with E.coli in ionic strengths of NH4Cl as shown in Figure 5.7. At the
ionic strengths of NH4Cl increased, dissociation of the salt distributed lots of free ions
in solution. Hence, excessive ions in solution would surpass the net negative charge on
S.aureus. As a result, lower electrophoretic mobility was obtained at higher ionic
strengths of NH4Cl solutions. Meanwhile, the magnitude of the electrophoretic mobility
of live S.aureus was observed to be constant at ionic strength of 0.01 M to 0.03 M. This
indicated that surface charge of live S.aureus was not affected much at this short range
of ionic strengths of solution. Comparatively, dead S.aureus decreased linearly at this
range of ionic strengths of NH4Cl solutions. This demonstrated that the surface charge
of dead S.aureus was affected even at lower ionic strengths of NH Cl solutions. 4
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.000.00 2.00 4.00 6.00 8.00 10.00 12.00
TiO2Live SADead SAPoly. (TiO2)Poly. (Live SA)Poly. (Dead SA)
1/ I
The
elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10-8
)
Figure 5.8. The magnitude of the electrophoretic mobility of TiO2, live and dead S.aureus versus the reciprocal of the square root of the ionic strength of NH4Cl. The dots with error bars are experimental points, the best-fit continuous and dashed lines were plotted with using second order of polynomial least square analysis.
As can be seen from Figure 5.8, the magnitude of the electrophoretic mobility of
dead S.aureus was obtained to be lower in negativity than live S.aureus at various ionic
strengths of NH4Cl solutions. This might due to dead S.aureus was metabolically
inactive and the protein composition in the cells might not strong bonded, hence
influence the net charge on surface.
The magnitude of the magnitude of the electrophoretic mobility of TiO2 at different
ionic strengths of NH4Cl was the highest compared to the live and dead S.aureus. This
was due to TiO2, being solid colloidal particles, whose movements were not impeded by
penetration of ions from the surrounding, as were biological cells with membranes. Also,
there was no ion exchange from the charged particles, stabilizing the negative surface
charge of TiO . 2
5.5.4 Effects of NH4Cl on the magnitude of the electrophoretic mobility of gram-positive and negative bacteria
The magnitude of the electrophoretic mobility of live S.aureus was more
negative than live E.coli at different ionic strengths of NH4Cl. This was because the
thicker peptidoglycan layer of S.aureus reduced the loss of ions from the interior cell
and the influx of ions from the medium. On the other hand, the thinner cell walls of
E.coli were more easily neutralized by the surrounding ions. Hence, the overall
electrophoretic mobility of E.coli was always less negative than S.aureus.
Cl on the surface charge of bacteria 5.5.5 Conclusion on the effects of NH4
Ammonium chloride will undergo completely dissociation in phosphate buffer
of pH 7 solution consequently increased conductivity of solutions as the ionic strength
of salt increased regardless of the types of suspension materials. This was due to the
same chemical environment shared among TiO2, live and dead E.coli and S.aureus. The
increase in the ionic strength of NH4Cl led to a decrease in the magnitude of the
electrophoretic mobility of TiO2, live and dead bacteria. This was because the increase
in ionic strength of NH4Cl increased free ions in solution, which might have overcome
the conductivity of the charged particles or cells when an electric field was applied. This
reduced the migration rate of charged particles and cells, leading to a lower magnitude
of electrophoretic mobility.
TiO2 is a solid particle that carries a negative surface charge and obtained the
highest magnitude of electrophoretic mobility in decreasing ionic strengths of NH4Cl
compared to the live and dead E.coli and S.aureus. Meanwhile, live S.aureus recorded
greater magnitude of electrophoretic mobility negativity than live E.coli. This might due
to the smaller size of S.aureus and more rigid cell wall of gram-positive S.aureus
compare to gram-negative E.coli, which is able to adsorb more positive ions on the
surface and increased the net negative charge of cells. On the other hand, dead E.coli
and S.aureus obtained a lower electrophoretic mobility at different concentrations of
NH4Cl than live E.coli and S.aureus. This indicated that the increase in permeability of
cell membrane and inactivate metabolism of dead cells screened by the surrounding ions
were unable to recover as live cells does, which leading to a lower negative surface
charge.
5.6 Effects of different ionic strength of sodium chloride (NaCl) on surface charge of biological cells and colloidal particles
Salt concentrations would lead to change of surface charge of cell membrane. In
addition, the structure of cell membrane could be changed as well in different
concentration of salinity. Hence, the effects of different ionic strengths of sodium
chloride (NaCl) on the surface charge of TiO , live and dead bacteria were investigated. 2
The increase in sodium chloride ionic strength exerts provides a greater pressure on
cells. The internal pressure is borne by the peptidoglycan layer and is maintained by
passive and active influx and efflux of ions from the surrounding liquid medium [109].
Thus, the sudden exposure of microorganisms to low or high ionic strength conditions
would disturb the balance of ion transfer and results in changes of the cell envelope
[125]. These changes might cause the microorganism to carry out modification to
achieve their stability in the suspension. However, modification can also result in
leakage of internal cell components such as periplasmic and cytoplasmic enzymes and
ions due to the high salinity solutions. The periplasmic space is the space between the
inner and outer plasma membranes of cell membrane bacteria. The substance that
occupies the periplasmic space is referred to as the periplasm which gram-positive
bacteria have a smaller periplasmic space. This space is involved in various biochemical
pathways including nutrient acquisition, synthesis of peptidoglycan, electron transport,
and alteration of substances toxic to the cell. Meanwhile, the cytoplasm is a gelatinous,
semi-transparent fluid that fills most of the cell. Eukaryotic cells contain a nucleus that
is kept separate from the cytoplasm by a double membrane layer. Hence, the
modification of cell in high salinity might lead to change in the inner structure of cells
which influence the net negative surface charge of bacteria.
NaCl as an ionic solute also influence the conformation and packing of lipid
headgroup of cells. Study showed that there was an increase in anionic lipids served to
give charge balance to the membrane surface when exposed to high Na+ concentrations
[126]. However, the extra amount of this extra lipid synthesized in a cell is enough to
account for only milimolar concentrations of NaCl. If the concentration of NaCl is too
high, the extra lipids are not able to balance the charge different of cells [127, 128].
Another theory account for the increase in anionic lipid content in high salinity is
due to the mechanism for preserving the membrane lipid bilayer. In high salinity
solution, the selective permeable system would collapse, and causing Na+ to influx into
cells and disrupt the metabolism since most intracellular enzymes of microorganisms
are inhibited by quite modest of increases in NaCl concentrations [129].
Besides, the increase in ionic strengths of NaCl elevates amount of free ions in
solution. This would lead to a greater conductivity of the solution with suspended
materials when an electric field was applied. In this study, samples were suspended in
phosphate buffer pH 7 solutions with NaCl ranging from 0.01 M to 0.08 M. The effects
of this salt concentration on electrophoretic mobility of TiO2, live, dead for both E.coli
and S.aureus was measured and studied.
5.6.1 Effects of different ionic strengths of NaCl on the conductivity of electrolyte
The influence of different NaCl concentrations on conductivity of solutions was
investigated. This study is important to gain information on the change of conductivity
of solutions with different suspended materials, as the conductivity might affect the
magnitude of the electrophoretic mobility of all the samples. For instance, the raise in
temperature due to high conductivity might change surface charges of samples.
Figure 5.9 shows the plot of molar conductivity, Λ versus samples in
different c , concentrations of NaCl indicated that the conductivity of solution
increased linearly with increased of ionic strengths regardless of suspended materials.
0
2
4
6
8
10
12
0.00 0.05 0.10 0.15 0.20 0.25 0.30
TiO2Live S.aureusDead S.aureusLive E.coliDead E.coli
Λ,
Mol
ar c
ondu
ctiv
ity
, m
S/cm
c , Concentration
Figure 5.9. Molar conductivity, Λ of TiO2, live and dead E.coli and S.aureus in different concentrations, c of NaCl.
The ion dissociation of NaCl increases the number of free ions in solutions.
Hence, the free ions increase the solution conductivity when an electric field is applied.
Conductivity of the solution with different suspended materials does not change very in
these ionic strengths of NaCl.
5.6.2 Effects of NaCl on the magnitude of the electrophoretic mobility of live and dead E.coli (TiO as reference sample) 2
Electrophoretic mobility of gram-negative E.coli in NaCl solutions was
investigated. Meanwhile, colloidal particle of TiO2 were used as a reference sample to
compare the result with E.coli and is shown in Figure 5.10. The magnitude of the
electrophoretic mobility of live and dead E.coli increased as the ionic strengths of NaCl
increased. This indicated that the net negative surface charge of E.coli increases as the
ionic strengths of NaCl increased. This might due to the increase in anionic lipids that
exist to give charge balance at membrane surface when expose to high concentration of
NaCl [126].
-4.00
-3.00
-2.00
-1.00
0.00
1.00
2.002.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
TiO2Live E.coliDead E.coliPoly. (TiO2)Poly. (Dead E.coli)Poly. (Live E.coli)
Figure 5.10. The magnitude of the electrophoretic mobility of live and dead E.coli (TiO2 as reference sample) versus the reciprocal of the square root of ionic strength of NaCl. The dots with error bar are the experimental points, the lines and dash lines of best fit are plotted with second order of polynomial least square analysis.
1/ I
The
elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10-8
)
Both live and dead E.coli did not differ much in their electrophoretic mobility
along the range of NaCl concentrations. This is due to the Na+ ions rush into the cells
and inhibit metabolism of live cells [127], which is similar with the metabolically
inactive dead E.coli. Consequently, the result of electrophoretic mobility of live and
dead E.coli was not much different.
However, the magnitude of the electrophoretic mobility of TiO2 was reversed
with E.coli in NaCl concentrations. For TiO2, the magnitude of the electrophoretic
mobility decreased at higher ionic strengths of NaCl as free ions increased the
compressibility of the ions on charged particles [130] as TiO2 is impenetrable to ions.
At higher ionic strengths, ions from the surrounding would migrate faster than the
charged particles when electric field applied. As a result, the magnitude of the
electrophoretic mobility of TiO2 as a solid particle was inversely proportional to the
increase in ionic strengths of NaCl.
5.6.3 Effects of NaCl on the magnitude of the electrophoretic mobility of live and dead S.aureus (TiO as reference sample) 2
The magnitude of the electrophoretic mobility of gram-positive S.aureus in
different concentration of NaCl was investigated and depicted in Figure 5.11. The result
of the magnitude of the electrophoretic mobility of TiO2 was used to compare with
S.aureus as non-biological samples.
Figure 5.11. The magnitude of the electrophoretic mobility of live and dead S.aureus (TiO2 as reference sample) versus the reciprocal of the square root for ionic strengths NaCl. Dots with error bars are experimental points. Solid and dashed lines of best fit are plotted with second order polynomial least squares analysis.
1/ I
The
elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
-4.00
-3.00
-2.00
-1.00
0.00
1.00
2.002.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
TiO2Live SADead SAPoly. (Live SA)Poly. (TiO2)Poly. (Dead SA)
The results show that the magnitude of the magnitude of the electrophoretic
mobility of live and S.aureus increases as the ionic strengths of NaCl increases. At low
concentrations of NaCl, positive electrophoretic mobility was obtained from both live
and dead S.aureus. This might due to the screen effects on negative charge of S.aureus.
However, as the concentration of NaCl increased, more Na+ ions from the solutions
disrupted the metabolism of live S.aureus. The live cells would increase the anionic
+ lipids to overcome the excessive influx of Na ions. As a consequent, the net negative
charge of cells increases and led to a more negative magnitude of electrophoretic
mobility at higher salinity. Besides, live S.aureus showed erratic electrophoretic
mobility at high concentration of NaCl. This showed that the thicker cell wall of
S.aureus resisted excessive Na+ ions to flow into cells. On the other hand, the magnitude
of the electrophoretic mobility of dead S.aureus decreased after 0.05 M until 0.01 M of
NaCl. The result of dead S.aureus demonstrated that the net negative surface charge of
S.aureus was affected at low ionic strengths of NaCl, but no changed on higher salinity.
As comparison with colloidal particle, TiO2 which is impenetrable to ions obeys
Equation 5.7. Hence, the results obtained from electrophoretic mobility of TiO2 are
opposite to that of live and dead S.aureus.
5.6.4 Effects of NaCl on the magnitude of the electrophoretic mobility of live
E.coli and S.aureus
The magnitude of the electrophoretic mobility of S.aureus and E.coli increased
with increasing ionic strength of NaCl. In comparison between both gram-positive and
–negative bacteria, live S.aureus demonstrated higher negative surface charges than live
E.coli in different concentration of NaCl. This might due to the greater size of E.coli
compare to S.aureus, which density of net negative charges is higher in smaller cells.
Another reason may be attributed to the thicker cell wall of S.aureus is able to prevent
extra Na+ ions to influx into the cells to cause change in lipids.
5.6.5 Conclusion of effects of NaCl
Both live and dead E.coli and S.aureus showed that the magnitude of the
electrophoretic mobility increased as salinity of solution increased. This was
contradicted with Equation 5.7, which indicated that the magnitude of the
electrophoretic mobility should be inversely proportional to the ionic strength of the salt.
This is because of the Na+ ions from the solution would influx into the cells. This
initiates the change of lipid to balance the inner charges and hence increase the anionic
lipids. As a consequent, the net negative charge of cells increases, and leads to a higher
electrophoretic mobility as concentration of NaCl increases. Unlike TiO2, colloidal
particle that is impenetrable to ions decreased in electrophoretic mobility as salinity
increased, which ions from the solution responded faster than the charged particle when
electric field was applied.
5.6.6 Donnan potential of live E.coli and S.aureus in various ionic strength of NaCl
The Donnan potential is a result of the Donnan equilibrium, which refers to the
distribution of ion species between two ionic solutions separated by selectivity
-permeable cell wall or boundary. This boundary layer maintains an unequal distribution
of ionic solute concentration by acting as a selective barrier to ionic diffusion. Some
species of ions may pass through the barrier while others may not. The solutions can be
gels or colloids as well as ionic liquids, and, as such, the phase boundary between gels
or a gel and a liquid can also act as a selective barrier. Electric potential arises between
two solutions is called the Donnan potential.
The electrophoresis system uses the Smoluchowski mobility formula which is
considered unsatisfactory for calculations of biological cell surface charge. The
Smoluchowski formula solely concerns with electric charges located only at
ion-impermeable particle surfaces of zero thickness. However, surface charges of
biological cells are distributed throughout an ion penetration layer of finite thickness [36,
110]. Hence, a new formula for the magnitude of the electrophoretic mobility of
biological cells was introduced by Ohshima [131]. The approximation of their mobility
formula depends on a weighted average of the Donnan potential and the potential at the
boundary between the surface charge layer and the surrounding medium.
Ohshima [131] electrokinetic theory of bacteria or “soft” particles considers a
spherical colloidal particle with a core diameter, a, coated with an ion penetrable layer
of polyelectrolytes with a thickness of d. The outer diameter is b = a+2d. Assumption is
made to suit the formula which includes fixing “soft” particle charge density, ρfix and
the dielectric permittivities of the liquid (εr) inside and outside the “soft” particle layer
are identical. The general equation for the magnitude of the electrophoretic mobility, μ,
is derived and based on the two parameters specific to the “soft” particle or bacteria.
The first parameter is the fixed charge density in the “soft” particles or bacteria
layer:
NZefix =ρ (5.8)
where N and Z are the number of concentration and the valence of the dissociated
functional groups in the “soft” particle layer respectively and e is the elementary electric
charge. Next, the parameter is related to the electrophoretic softness, 1/λ, given in length
units. It is defined as:
2/11
⎟⎟⎠
⎞⎜⎜⎝
⎛=
γη
λ (5.9)
where η is the viscosity of the medium and γ is the frictional coefficient of the surface
charge layer. For cases where кa/2>>1, a/2>>d, λd>>1 and кd>>1, with к being the
Debye-Hückel parameter and when the relaxation effect of the electrical field on the
particle double layer is neglected, then the magnitude of the electrophoretic mobility of
the soft particle is given by [110, 36, 132].
20
/1/1//
ηλρ
λκλψκψ
ηεεμ fix
m
DONmor +++
= (5.10)
with the outer surface potential Ψo, the Donnan potential in the “soft” particle layer
Ψ , and the Debye- Hückel parameter of surface region, кm , given as follows: DON
⎟⎟⎟
⎠
⎞
⎜⎜⎜
⎝
⎛
⎥⎥⎥
⎦
⎤
⎢⎢⎢
⎣
⎡
⎪⎭
⎪⎬⎫
⎪⎩
⎪⎨⎧
+⎟⎟⎠
⎞⎜⎜⎝
⎛−+
⎥⎥⎥
⎦
⎤
⎢⎢⎢
⎣
⎡
⎪⎭
⎪⎬⎫
⎪⎩
⎪⎨⎧
+⎟⎟⎠
⎞⎜⎜⎝
⎛+=
2/122/12
12
12122
lnzen
zenzenzenze
kT fix
fix
fixfixo
ρρ
ρρψ (5.11)
⎟⎟⎟
⎠
⎞
⎜⎜⎜
⎝
⎛
⎥⎥⎥
⎦
⎤
⎢⎢⎢
⎣
⎡
⎪⎭
⎪⎬⎫
⎪⎩
⎪⎨⎧
+⎟⎟⎠
⎞⎜⎜⎝
⎛+=
2/12
122
lnzenzenze
kT fixfixDON
ρρψ (5.12)
4/12
21
⎥⎥⎦
⎤
⎢⎢⎣
⎡⎟⎟⎠
⎞⎜⎜⎝
⎛+=
zenfix
m
ρκκ (5.13)
2/1222
⎟⎟⎠
⎞⎜⎜⎝
⎛=
or
enzεε
κ (5.14)
εo denotes permittivity in a vacuum, k is the Boltmann constant, T is the absolute
temperature, z and n are the valence and bulk number concentration of the symmetrical
electrolyte in solution respectively.
A curve-fitting procedure was applied to the experimental values of eletrophoretic
mobility as a function of electrolyte concentration to determine the electrophoretic
softness, 1/λ, and fixed charge density, ρfix of the bacteria cells.
Equation 5.10 was utilized to simulate the magnitude of the electrophoretic
mobility values and compared with the actual measurement results. The magnitude of
the electrophoretic mobility as a function of ionic strength (NaCl) for two bacterial
strains is demonstrated is in Figures 5.12 and 5.13. The two strains showed negative
electrophoretic mobility, indicating that their net negative surface charge originated
from acidic functional groups on transmembrane proteins. As seen from Figures 5.9 and
5.10, Ohshima approximation curve showed that the magnitude of the electrophoretic
mobility of both bacterial strains became less negative as ionic strength increased. This
might attribute to the charge screened by counterions and compressed of ions at
electrical double layer [132]. On the other hand, the curve fitted well with the
experimental values after 0.05 M. This might due to the lower the ionic strength of
NaCl might have less effect on the surface charge of bacterial [133, 119, 134].
Figure 5.12. The magnitude of the electrophoretic mobility of ionic strength (NaCl) for E.coli at pH 7 (curve fit with Ohshima approximation and experimental result).
Ionic strength, M El
ectro
phor
etic
mob
ility
, (x
108 ) m
2 V-1
s-1
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.00
0.500 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
Numerical value approximation(Oshima)Practical points E.ColiPoly. (Numerical value approximation(Oshima))
Figure 5.13. The magnitude of the electrophoretic mobility of ionic strength (NaCl) for S.aureus at pH 7 (curve fit with Ohshima approximation and experimental result).
Ionic strength, M
Elec
troph
oret
ic m
obili
ty ,
(x10
8 ) m2 V
-1s-1
-10.00
-8.00
2.00
-6.00
-4.00
-2.00
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
0.00
Numerical approximation (Oshima)Practical points of S.APoly. (Numerical approximation (Oshima))
The fixed charge density, ρfix for E.coli and S.aureus was -115 mM and -155.6
mM respectively. Meanwhile, the electrophoretic softness, 1/λ, for E.coli and S.aureus
was 0.5 nm and 1.5 nm respectively. Besides, different culturing media is also affecting
the electrophoretic softness of the bacteria [123]. If the eletrophoretic softness is large,
it means that the cell surface is hard and vice versa [18]. In this study, results showed
that the surface layer of S.aureus was harder than E.coli.
5.6.7 Conclusion on curve fitting of Donnan potential
Study reported that the Smoluchowski formula is solely concerning the electric
charges that located at the ion impermeable particle surfaces with zero thickness.
However, for biological cells, surface charges are distributed through ion penetration
layer of finite thickness [36, 110] . Hence, the Donnan potential is introduced to
calculate the magnitude of the electrophoretic mobility of biological cells [131]. In this
study, the Ohshima’s numerical approximation for electrophoretic mobility fitted well
with live E.coli and S.aureus at higher ionic strength, which approximately from 0.05 M
onwards. This might due to unstable polarization of ions and cells or impurities of
solutions that affected the result at lower ionic strength of NaCl.
5.7 Summary on effects of the magnitude of the electrophoretic mobility in different chemical environment
In this chapter, the surface charge of bacteria and TiO2 in different chemical
conditions was investigated. Study showed that increase in washing increased the
magnitude of the electrophoretic mobility of bacteria. On the other hand, E.coli
recorded the lowest magnitude of the magnitude of the electrophoretic mobility in
distilled water, pH, NaCl and NH Cl as compared to S.aureus and TiO4 2. This could be
due to the larger size of E.coli than other samples. In various pH, dead cells of both
E.coli and S.aureus obtained the lower magnitude of the magnitude of the
electrophoretic mobility than live cells. This was mainly due to dead cell had lost the
metabolic activity to give charge balance to surface charges. The increased in pH
increased the magnitude of the electrophoretic mobility of bacteria and TiO2, which was
due to the higher concentration of hydroxyl groups, negative ions in alkaline solutions
increased the net surface charge of the samples. Besides, it was found that the increase
in ionic strengths of NH4Cl reduced the magnitude of the electrophoretic mobility of
samples. This is because of the share of current carried by bacteria and particles
decreased as ions in solutions increased. In contrary, increased ionic strengths of NaCl
obtained the opposite result for bacteria. This might due to increase of influx Na+ into
cells increase the anionic lipid to balance the cells. As a result, this led to greater net
negative surface charge. In addition, the magnitude of the electrophoretic mobility of
live bacteria in NaCl was compared with the numerical approximation with the Donnan
potential approximation. Result indicated that both approached fitted well at greater
ionic strength of NaCl.
The negative surface charge of S.aureus was higher than E.coli for all different
study conditions. The affecting factors might due to the smaller size of S.aureus possess
greater density of net negative charge on surface. However, the main different of these
two strains of bacteria is the thickness of peptidoglycan layer, which gram-positive
S.aureus has thicker cell wall than E.coli. Generally, TiO2 recorded the highest negative
magnitude of the magnitude of the electrophoretic mobility compares to bacteria. This
may due to no metabolic activity occurs in solid particles, hence the net negative charge
on surface is more rigid.
CHAPTER 6
Effects of Temperature and Applied Field on Electrophoretic Mobility
6.0 Effects of temperature on the magnitude of the electrophoretic mobility of colloidal particles and bacteria
Temperature is an essential parameter to determine the stability of colloidal
suspension. Temperature affects the crystallization of TiO2 [135] , photocatalytic
activity and film adhesion [136, 31]. Study showed that liposome structural action
formation was also affected by temperature [137]. A report on the phase transition
temperature of liposome lipid showed that the phosphatidyl group lies in the outer-most
region of the surface and the choline group is in the inner-most region [138].
Consequently, the structure of liposome might change due to the elevation in
temperature and this changed the properties of the surface charge. Besides, it was
reported that the ability of biological cells to withstand the change of temperature
depends on fluidity of cells [127].
There is essential concept on understanding temperature elevation, which the
increase in temperature increases kinetic energy of particles and ions in solution. This
causes counterions on the charged particles or cells thickening as increase in diffusion.
This leads to loose adhesion of opposite ions to double diffuse layer. Thereby, higher
magnitude of the magnitude of the electrophoretic mobility was obtained as temperature
increased. Besides, temperature elevation also reduces viscosity of solution, this
decreases the frictional forces among the suspended particles and cells. As a result,
charged particles and cells are able to respond faster as electric field was applied [89].
Since temperature plays an important role in affecting surface charges, hence this
parameter was used to study the change of surface charges of colloidal particles, TiO2
and liposome, gram-positive and gram-negative bacteria.
The Hückel–Onsager and Helmholtz-Smoluchowski shows in Equations 3.5 and
3.6 respectively were calculated to predict the magnitude of the electrophoretic mobility
of TiO2 and liposome. Meanwhile, numerical result was used to fit experimental results
for both samples. In this calculation, zeta potential was assumed to be fixed at -45 mV.
On the other hand, changes of solution viscosity at various temperatures were taken
from CRC handbook of Chemistry and Physics [43]. Besides, solution permittivity was
assumed to be unchanged at 80. f(κa) is a function of the particle size (radius a), with
value of 1.5. Lastly, 1/k represents the thickness of the double layer of counter ions.
6.1 Comparison of theoretical and experimental electrophoretic mobility of TiO2 and liposome
Temperature elevation reduces viscosity of solution. At lower viscosity,
frictional forces of ions in solution decreases. As a result, higher electrophoretic
mobility of suspended materials will be obtained. In this study, the equations of
Hückel–Onsager and Helmholtz-Smoluchowski were calculated to predict the
magnitude of the electrophoretic mobility with the changes of viscosity due to the
increase in temperature. The predicted result latter fitted with experimental result of
electrophoretic mobility of TiO and liposome2 . This is to investigate the most suitable
equations for electrophoretic mobility calculation. Hence, the predicted result of
electrophoretic mobility with Hückel–Onsager and Helmholtz-Smoluchowski equation
compared with experimental result of colloidal particles are shown in Figure 6.1 and
6.2.
The predicted magnitude of the electrophoretic mobilities at various
temperatures using the Hückel–Onsager equation was found lower than those calculated
from the Helmholtz-Smoluchowski equation. There was slightly difference between
these formulae, which the Helmholtz-Smoluchowski equation assumes the Debye length
constant, f(ka) as 1.5.
The magnitude of the electrophoretic mobility measured for TiO2 fitted better to
the Helmholtz-Smoluchowski and was not too discrepant from the Hückel–Onsager
curve.
Temperature, 0C
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
-5.00
-4.50
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.000 10 20 30 40 50 60
Helmholtz-Smoluchowski Theory lineHuckel Onsager Theory LineTiO2 Practical result
Figure 6.1. Hückel–Onsager and Helmholtz-Smoluchowski predicted electrophoretic mobility values compared with experimental values of TiO2 in a range of temperatures.
Figure 6.1 indicates that the magnitude of the electrophoretic mobility of TiO2
increases as temperature increased. This demonstrates that the net negative charges on
surface of TiO2 increases in higher temperature aqueous system. This is because, higher
temperature increases kinetic energy of ions in solution, which diffuse the counterions
on surface of charged particles and thickening the double diffuse layer. Hence, higher
electrophoretic mobility was obtained [139].
On the other hand, Figure 6.2 shows the experimental result of liposome in a range
of temperatures fitted well to the Helmholtz-Smoluchowski curve. The magnitude of the
electrophoretic mobility displayed minor discrepancy from the curve at higher
temperatures might due to the disturbance from minor impurities in the solution.
Besides, the magnitude of the electrophoretic mobility of liposome increased in the
range of temperatures indicated that the greater kinetic energy absorbed by ions and
charged particles reduced viscosity of solution and responded faster to electrostatic
forces on each other and move faster as electric field was applied. As a consequent,
higher the magnitude of the electrophoretic mobility of liposome as temperature
increased.
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.000 10 20 30 40 50 60
Huckel Onsager theoryHelmholtz-Smoluchowski theoryLiposome Experimental
Temperature, 0C
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
Figure 6.2. Hückel–Onsager and Helmholtz-Smoluchowski predicted electrophoretic mobility values compared with experimental values of liposome in a range of temperatures.
The Hückel–Onsager and Helmholtz-Smoluchowski numerical curves together with
the experimental results of colloidal particles, TiO2 and liposome indicated that the
Helmholtz-Smoluchowski equation was suitable to be used to calculate electrophoretic
mobility of the charged particles.
6.1.1 Comparison of zeta potential of colloidal particles at the range of temperatures
The magnitude of the electrophoretic mobility, which was straightly calculated
from velocity of charged particles when electric field was applied, was compared with
zeta potential that was predicted from formula of the Helmholtz-Smoluchowski to
estimate density of counterions on surface at a range of temperatures.
Figure 6.3. The comparison of zeta potential between liposome and TiO2.
-30.0
-25.0
-20.0
-15.0
-10.0
-5.0
0.0
Temperature, oC
0 10 20 30 40 50 60
Zeta
pot
entia
l, m
V
LiposomeTiO2
Figure 6.3 shows zeta potential of TiO2 and liposome. Both result indicated that
the increase in temperatures was not propotional to zeta potential. This contradicted
with the magnitude of the electrophoretic mobility, which the increased of temperatures
increased the magnitude of the electrophoretic mobility. The negativity of zeta potential
decreased as temperature increased from 20 oC to 40 oC. Since the zeta potential is
estimated from the difference of electrokinetic potential between the tightly bound
layers around the surface till the diffuse layer and depended also on the density of
counterions around the charged particles, thereby, as temperature increased, the ions in
the diffuse layer diffuse greatly into the surrounding solution and moved more randomly
and away from the charged particles. Furthermore, the opposite ions in the compact
layer which were immobile due to being tightly bound to the surface particles initially,
but might start to diffuse because of the increase in thermal energy absorption.
Consequently, this reduces the zeta potential. In contrary, the net negative charges on
the surface of TiO2 increased as the screen effect decreased. This led to a more
significant migrating rate towards the electrode when an electric field was applied and
higher the magnitude of the electrophoretic mobility was obtained. The negative zeta
potential of liposome was greater than those of TiO2. The smaller size of liposome is
account for the faster mobility than TiO . 2
6.1.2 Comparison of the magnitude of the electrophoretic mobility of TiO2 and liposome at the range of temperature
Colloidal particles, TiO2 and liposome was compared the magnitude of the
electrophoretic mobility at different temperatures. The Helmholtz-Smoluchowski
equation was also calculated to predict the magnitude of the electrophoretic mobility of
TiO2 and liposome, which can be seen in Figure 6.5. The electrophoretic mobility of
liposome fitted well with predicted curve compared to TiO2. Meanwhile, electrophoretic
mobility of TiO2 fitted better with predicted curve from 20 o oC to 35 C and deviated
slightly from 40 o oC to 50 C.
Temperature, 0C
0.00
Figure 6.4. Actual and predicted electrophoretic mobility of TiO2 and liposome at various temperatures and the predicted curve was based on the Helmholtz-Smoluchowski equation.
Elec
troph
oret
ic m
obili
ty, m
2 V
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0 10 20 30 40 50 60
The actual experimental electrophoretic mobility of liposome appeared to be greater
than that of TiO2, as can be depicted in Figure 6.4. The magnitude of the electrophoretic
mobility of TiO2 increased from -2.63 × 10-8 m2 V-1 s-1 to -4.46 × 10-8 m2 V-1 s-1.
Meanwhile, the magnitude of the electrophoretic mobility of liposome increased from
-3.29 × 10-8 m2 V-1 s-1 to -5.45 × 10-8 m2 V-1 s-1. The total change in the magnitude of the
-1-1 s
(x 1
0 )
-8
Theoretical line for TiO2TiO2LiposomeTheoretical line for liposome
electrophoretic mobility throughout the range of temperatures was higher for liposome.
It has been demonstrated that the negative surface charge of liposome was greater than
TiO . This is because of the semi-solid liposome and smaller in size than TiO2 2.
Consequently, a smaller amount of heat absorption is sufficient to increase the kinetic
energy of particles to migrate towards the electrode. However, the stability of liposome
is dependent on the temperature. If liposome exposes to higher temperatures would have
a higher propensity of its lipids undergoing a transition to a non-bilayer phase and this
might cause the oxidative deterioration of liposome phospholipid which influences
liposome stability [140]. Since the highest temperature in this study was 55 oC, hence
this range of temperature was not sufficiently high to cause changes on structure of
liposome.
6.1.3 Electrophoretic mobility live and dead Escherichia coli at a range of temperature (TiO as reference sample) 2
Effect of temperatures on the magnitude of the electrophoretic mobility of live
and dead E.coli, together with TiO2 as a non-biological sample was studied and shown
in Figure 6.6. The magnitude of the electrophoretic mobility of live E.coli showed a
mild increased as temperature increased. The difference between the highest and the
lowest magnitude of the electrophoretic mobility values of live E.coli was only a
marginal of -0.53 × 10-8 m2 V-1 -1s . This result was similar to that of TiO2 in this range
of temperatures. The only different was that, TiO2 possessed a much higher
electrophoretic mobility magnitude than E.coli. As a result, both the magnitude of the
electrophoretic mobility of TiO2 and live E.coli increased as temperatures elevated. This
is due to the increase in temperatures reduce viscosity of solution. The energetic ions
and charged particles or cells were able to move faster when electric field was applied.
Another reason for the higher negative surface charge of TiO than E.coli might due to 2
smaller in size of this solid particles, TiO2 was able to migrate faster under applied field,
thus causing a higher magnitude of the electrophoretic mobility. Meanwhile, live E.coli
was assumed to be able to resist the change on its surface charge. That is the
explaination on the mild increase in the magnitude of the electrophoretic mobility at this
range of temperatures.
Figure 6.5. Effects of temperature (20oC-55oC) on electrophoretic mobility of TiO2, live and dead E.coli (Electrophoretic mobility of TiO2 measured is help to guide the trend as temperature increased).
Temperature (oC)
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10-8
)
-5.00
-4.50
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.000 10 20 30 40 50 60
Live E.ColiDead E.ColiTiO2
In Figure 6.5, dead E.coli indicated the most erratic result compared with live
E.coli and TiO o o. From 25 C to 352 C, the magnitude of the electrophoretic mobility of
dead E.coli displayed a sharp decrease in magnitude of the electrophoretic mobility
from -2.11 × 10-8 m2 -1 -1 -8 V s to -1.24 × 10 m2 -1V s-1, which gave a difference of -0.87 ×
10-8 m2 V-1 -1s . The negative magnitude of the electrophoretic mobility of dead E.coli
increased at 40 oC but reduced for the next three increased of temperatures. Factor that
brought to the erratic result of dead E.coli might due to the increase in permeability of
ions into cells that disrupt the balance of charges on surface. The amount of fluid inside
dead cells would change, hence unable to respond to the change in temperatures.
Another reason for the erractic result of dead E.coli may be attributed to the protein of
the cells starts to decompose as temperature increases.
The negative surface charge of E.coli is due to the surface layer which is
associated to the lipopolysacharide and ions with protein carbohydrates interactions or
protein-protein interactions [61]. Carboxyl functional groups attached to the proteins
groups associated with teichoic acids can deprotonate to form negatively charge metal
binding sites. These anionic functional groups generate charge of the cell wall may
result in the formation of an electric field that surround the entire cell [12]. This has
lead to the negative electrophoretic mobility of live and dead E.coli measured.
The dead E.coli was obtained to be more electronegative compare to live E.coli
throughout the range of temperatures. It is due to the concentration of proton outside the
plasma membrane and the bound protein transports of ion sodium and ion hydrogen,
giving rise to a less negative net cell wall charge [120, 141]. It was reported that dead
intact cells would generate more effective negative surface charge [120].
On the other hand, a live biological cell has the ability to rebalance the ions in
and out from the membranes in order to and maintain the viability of the cell. The
average resting potential of a cell is -73 mV [142]. Resting potential are the amount of
surface charge that the cells has to maintain, which the cells keep on working to
neutralize the charges around the membrane cell with pumping ions in and out
continuously. This explains the marginal change of live cells in temperatures.
The corresponding zeta potential of live and dead E.coli were obtained and
depicted in Figure 6.6 for different temperatures. Although dead E.coli showed
erratically measured of zeta potential throughout the temperatures, but the zeta potential
for both live and dead E.coli was seen to have slight decreased in negativity as
temperature increased.
Figure 6.6. Zeta potential of live and dead E.coli corresponded to the magnitude of electrophoretic mobility that obtained from measurements.
Temperature (oC)
Zeta
Pot
entia
l (m
V)
-18
-16
-14
-12
-10
-8
-6
-4
-2
00 10 20 30 40 50 60
Live E.coli Dead E.coli
The decrease in zeta potential of E.coli as temperature increased. This is because of
the diffusion of counterions from the surface of cells. The thickening of double diffuse
layer measured a lower zeta potential as temperature increased.
6.1.4 Electrophoretic mobility live and dead S.aureus at a range of temperature (TiO as reference sample) 2
The magnitude of the electrophoretic mobility of gram-positive S.aureus which
possesses a thicker peptidoglycan layer was compared with E.coli at a range of
temperatures. As shown in Figure 6.7, the magnitude of the electrophoretic mobility of
live and dead S.aureus with TiO2 as reference sample was obtained. The magnitude of
the electrophoretic mobility of live and dead S.aureus increases slightly as temperature
increases. This indicated that the surface charge of S.aureus was not much affected in
this temperature range. Although the increase of the magnitude of the electrophoretic
mobility of S.aureus was not obvious in this range of temperature, but it showed that the
elevation of temperature increased the heat absorption of ions. The thickening of double
diffuse layer of S.aureus reduced the screening effect on the surface, which led to a
higher magnitude of the electrophoretic mobility.
-5.00
-4.50
-4.00
-3.50
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.000 10 20 30 40 50 60
TiO2Dead S.aureusLive S.aureus
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10-8
)
Temperature (oC)
Figure 6.7. Effects of temperature (20oC-55oC) on electrophoretic mobility of TiO2, live and dead S.aureus (Electrophoretic mobility of TiO2 measured is help to guide the trend as temperature increased).
The magnitude of the electrophoretic mobility of dead S.aureus was consistent
from 20 oC to 40 oC but increase in negative magnitude of the electrophoretic mobility
until the end of temperature range. This showed that the migration rate for dead
S.aureus increased at higher temperatures. The dead S.aureus was metabolically inactive
and lipids in the cells might be disrupted and shrink. This reduced the fluidity volume of
cells, hence the cell density was decreased. As a consequent, the electrophoretic
mobility of dead S.aureus increased at higher temperatures. This could also explain the
more negative electrophoretic mobility and zeta potential of dead S.aureus compared to
that of live S.aureus at higher temperatures, as can be seen in Figure 6.7 and 6.8
Figure 6.8. Zeta potential of live and dead S.aureus, which corresponded to the magnitude of electrophoretic mobility.
Zeta
pot
entia
l (m
V)
Temperature (oC)
-18
-16
-14
-12
-10
-8
-6
-4
-2
00 10 20 30 40 50 60
Dead S.aureusLive S.aureus
The corresponding zeta potential to magnitude of the electrophoretic mobility
measured was calculated to estimate the surface charge potential for this range of
temperature show in Figure 6.9. The data displays a gradual decrease in negative zeta
potential for both live and dead S.aureus as temperature increases. The changes of zeta
potential for both groups of S.aureus are ranging from -30 mV to -20 mV. The decrease
in zeta potential indicated that the double diffusion layer was thickening as temperature
increased. Hence, the potential difference between compact layer and diffuse layer was
lessening. Another alternative explanation on this is caused by the lowering of viscosity
of solution as temperature increased. In this situation, ions in solutions gained thermal
energy and led to increase in kinetic energy Thus, ions moved faster towards the
opposite attraction of negatively-charged cells and screened the negative surface charge
of cells. Thereby, the negativity of zeta potential decreased as temperature increased.
However, lowering the viscosity of solution reduced the frictional forces of cells when
electric field applied, which causing an increase in the magnitude of the electrophoretic
mobility as temperature elevated and this result can be observed from Figure 6.7 and
6.8.
6.1.5 Comparison of the magnitude of the electrophoretic mobility of gram positive and gram negative bacteria at range of temperatures
The magnitude of the electrophoretic mobility and corresponding zeta potential
of live gram-negative E.coli and gram-positive S.aureus at the same range of
temperatures were studied, as depicted in Figures 6.9 and 6.10 respectively. Figure 6.9
shows the decrease in the magnitude of the electrophoretic mobility for E.coli and
S.aureus, but indicated a gradual decreased in the corresponding zeta potential at the
determined range temperatures. Besides, result demonstrated that live E.coli was less
electronegative compared to live S.aureus which can be seen from both figures.
Temperature (oC)
Figure 6.10. Zeta potential measured of E.coli and S.aureus corresponded to the magnitude of the electrophoretic mobility.
Zeta
pot
entia
l (m
V)
Temperature (oC)
-16
-14
-12
-10
-8
-6
-4
-2
00 10 20 30 40 50 60
Live S.aureusLive E.coli
Figure 6.9. Effects of temperature (20oC-55oC) on electrophoretic mobility of E.coli and S.aureus.
Elec
troph
o
-3.00
-2.50
-2.00
-1.50
-1.00
-0.50
0010 20 30 40 50 60
0.0
retic
mob
ility
2 V-1
-1 (x
-8
10)
s, m
Live E.coliLive S.aureus
The increase in the magnitude of the electrophoretic mobility was due to the fast
moving ions and cells as viscosity of solution decreased at higher temperatures. This
phenomenon led to a lower magnitude of zeta potential, which was because the screens
effect was greater due to the lower frictional forces among cells and ions. Thus, the fast
moving ions at high temperatures were able to screen out the negative charged of cells
in a shorter time. In addition, the decrease in corresponding zeta potential might also be
due to the change of cells structure or decrease in anionic lipids as temperature
increased. Hence, the net negative charges on live cells reduced.
Live E.coli indicated a lower negative surface charge than S.aureus at the range
of temperatures. This might due to the different thickness of cell wall for both bacteria,
which was discussed in Chapter 2. Gram-negative E.coli comprises a thinner
peptidoglycan layer at cell wall but constitutes more complex proteins integrity than
gram positive S.aureus [61]. Thereby, negative surface charge of E.coli formed from the
proteins was easier to be screened by oppositely-charged ions due to the thinner cell
wall. In contrary, S.aureus with thicker cell wall reduced the diffusion rate of ions from
it. Therefore, it produced a more constant result throughout change of temperatures and
also obtained higher negative magnitude of the electrophoretic mobility than E.coli.
6.2 Effects of applied field on the magnitude of the electrophoretic mobility of colloidal particles and bacteria
It was reported that an increase in applied field decreases migration time of the
charged particles to electrode [143]. This leads to a greater velocity of the charged
particles and hence, increases the magnitude of the electrophoretic mobility. This might
due to the increase in applied field elevates the temperature of a solution which in turn,
affects the migration rate of charged particles to the electrode. The increase in
temperatures called Joule Heating effects and is generated by an electric current passes
through the solution within the capillary, causing an increase in temperature [144, 145].
Besides, the elevated temperature results in an increase in carrier electrolyte
conductivity and the solute diffusion coefficient. The increase in diffusion coefficient of
solution thickens the electrical double layer of charged particles, which then decreases
carrier density and the viscosity of solution [146]. Consequently, the increase in applied
field induces temperature elevation which leads to the reduction of viscosity in the
electrolyte solution. This predominantly accounts for the increase in electrophoretic
mobility [125]. Meanwhile, the thermal effect can be evaluated by Ohm’s plot (the plot
of current against the voltage gradient) based on the deviation from linearity.
Viscosity of solution reduces as the temperature increases and the relation with
electrophoretic mobility is shown in Equation 6.1 [108].
( )( )T
kafe η
ςεμ3
2= (6.1)
where η (T) is viscosity and T is absolute temperature, ε denotes the dielectric constant
of the dispersing medium, f(ka) is the function of particle size (radius, a), where 1/k is
the thickness of the double layer of counter ions and ions that surround an individual
particle.
Ohm’s law is essential to be employed in applying different voltages to a system as
current increases when an electrical field is applied. The size of current is also
determined by the resistance of medium and is proportional to voltages. The current in
solution between the electrodes is conducted mainly by buffer ions and charged
particles. An increase in voltages will increase the total charge per second conveyed
towards the electrode.
The effects of varying the applied field on electrophoretic mobility of samples were
investigated. According to Ohm’s law, voltage (V) increases the current (i), hence,
creating power dissipation which is referred to as Joule Heating. The total amount of
heat generated can be calculated using Equation 6.2 [99]:
( )L
AtVR
tVRtiiVttJ σ222 ==== (6.2)
where J is amount of heat (Joules) as a function of time, t (s), σ is conductivity of the
electrolyte, R is resistance when a potential applied across a capillary, A represents area
of cross-section and L is the length, of capillary.
In this study, the measurement duration was very short (2.6 ms) for each
different applied field, especially since equilibrium between heat generation and
dissipation to the surroundings had been established. Consequently, time would have no
influence on the magnitude of the electrophoretic mobility [99].
An increase in temperature reduces the resistance of the medium as ions in solution
move faster with absorption of thermal energy. The effects of Joule heating have been
modeled to predict the temperature inside a capillary [147, 148]. The temperature
difference, ΔT, is defined as follows:
2
2
LAV
T o
πχσ
=Δ (6.3)
where σ is the conductivity measured at the reference temperature (25oo C) for each
different sample , χ denotes thermal conductivity of the buffer and is taken as 0.561 W
m-1 -1 and V is the applied field. K
Equation 6.3 demonstrates that the increase in applied field elevates the
temperature of the solution. This phenomenon enables charged particles, cells and ions
to gain more internal energy to move faster towards the respective electrode with less
frictional forces. Meanwhile, counterions crowd at surface of charged particles or cells
will diffuse more if temperature increased. Consequently, higher electrophoretic
mobility can be obtained.
Conductivity of solution with the same ionic strength should not vary much for the
increase in electrical conductivity while temperature increases [149]. This can be seen
in Equation 6.4. However, the suspended materials in the same concentration of solution
might affect the magnitude of the electrophoretic mobility.
( )[ ]00 1 TT −+= ασσ (6.4)
where α is the temperature coefficient of conductivity, To is the reference temperature,
σ is the electrolyte conductivity and σo is the electrolyte conductivity at reference
temperature. The changed in electrophoretic mobility of colloidal particles and bacteria
measured due to varying field applied is discussed in the section below.
6.2.1 Ohm’s plot for colloidal particles and bacteria at different applied field
Ohm’s plot for colloidal particles and bacteria indicates that the increase in applied
field increases current measured in solution with suspended materials, and is depicted in
Figure 6.10. Result indicated that both dead E.coli and S.aureus obtained the greatest
increase in current, with a total increase of 33 (±1) mA and 34 (±1) mA respectively for
the whole range of applied fields. Furthermore, live E.coli and S.aureus demonstrated a
greater increase in current compared to colloidal particles, TiO2 and liposome. As
compare with both colloidal particles, the increase in current for liposome was higher
than TiO . For TiO2 2, current of solution increased for the range of applied field was
only 9 (±1) mA.
The obvious different of the current increased with applied fields of both biological
cells and colloidal particles, is due to live cells are semi-liquid particles which are less
dense than solid particles, providing more freedom for ions in solution to move towards
an electrode when an electric field is applied. The degree of freedom for ion and particle
movement increases as applied field increases, which in turn, enhances greater electric
field attraction of charged ions and particles to electrodes.
Cur
rent
(mA
)
Result from Figure 6.11 also shows that the higher current obtained from dead
bacteria compared to live bacteria and colloidal particles. This indicated that the
permeability of the ions from the solutions through dead cells increased as the metabolic
activity of the dead cells ceased. Live bacteria, which were metabolically active blocked
excessive ion flow across, thus, cells increasing the resistance of ion penetration.
Consequently, a lower increase in current was obtained compared to dead bacteria. On
the other hand, liposomes as semi-solid colloidal particles consists of simple structure of
live cells but with no metabolic activity, possesses a rather rigid outer layer of
membrane compared to biological cells. Hence, liposome obtained an increase in
current throughout the applied field but was lesser than those of dead and live biological
cells. In contrary to those semi-permeable suspended materials, TiO2 as a solid particle
demonstrated great resistance towards ions moving in a straight path towards electrodes.
This solid particle reduced the hydrodynamic movement of ions, and recorded the
-5.00
0.00
5.00
10.00
15.00
20.00
00
00
00
00
00
0 20 40 60 80 100 120 140
45.
40.
35.
30.
25.
TiO2LiposomeLive E.ColiDead E.ColiLive S.ADead S.A
Applied Voltage (V)
Figure 6.11. Ohm’s plot for colloidal particles and bacteria in varied applied field.
lowest increase in current within the range of applied field. The bacterial cell is less
dense compared to colloidal particles, consequently can move faster as applied field
increases in the electrolyte.
6.2.2 Temperature changes against applied fields due to current increase
The increase in applied field increased the current of solution when an electric
field was supplied. This led to an elevation of temperatures of solutions, as can be
observed from Equation 6.3, which demonstrates the proportional relationship between
applied field and temperature difference. Figure 6.12 shows the relation between
applied field and total temperature changes in colloidal particles and bacteria. Results
indicated that a slower and more gradual changes in lower temperature at lower applied
field but more drastic increase after 80 oC. Besides, temperatures for all samples
increased at the same rate from 15 o oC to 40 C but started to display differences at
higher temperatures, which was due to the higher applied voltage was supplied.
Among the samples, greater changes in temperature for the applied field range
were shown in dead S.aureus. In contrary, the lowest changes in temperature as applied
field increased were obtained from TiO2. Meanwhile, total difference in temperature
changed throughout the range of applied field was 13 oC with dead S.aureus, which
obtained a higher temperature different of 10 oC compared to TiO2. On the other hand,
live, dead E.coli and S.aureus demonstrated greater differences in temperature
compared to colloidal particles.
20
25
30
35
40
45
50
55
0 20 40 60 80 100 120 14
0
TiO2LiposomeLive E.ColiDead E.ColiLive S.ADead S.A
Voltage , V
Tem
pera
ture
, o C
Figure 6.12. Temperature changes when applied field increase for colloidal particles and bacteria
The increase in current that passed through the solution when an electric field was
applied induced temperature changes in the electrolyte. This is due to the faster
migration rate of ions and charged particles under an increase of electric field. At lower
applied field, the elevation of temperature was not obvious but increased drastically at
higher voltages. This meant that higher applied fields caused greater heat dissipation of
the electrolyte. Besides, the data showed that biological cells had a higher temperature
elevation compared to colloidal particles, thus, collaborating the results shown by
Ohm’s plot. This showed that the less dense biological particles heated up faster than
colloidal particles, especially TiO2 which were able to absorb more heat. Meanwhile,
liposome did not exhibit a greater temperature change as compared to biological cells.
This demonstrated that liposome tolerated heat more than live and dead bacteria.
Table 6.1 Temperature coefficients, α is calculated for colloidal particles and bacteria.
In a solution, temperature coefficient is a measure of change in conductivity which
brought by the change of temperature. Temperature coefficient was estimated with
Equation 5.3 and shows in Table 6.1. It showed the highest temperature coefficient
obtained from TiO2 and liposome. This was followed by the dead population of bacteria.
The dead bacteria gave a higher temperature coefficient than live populations of bacteria.
The overall calculation showed that conductivity of solution suspended with colloidal
particles was higher than the temperature changes of the biological cells.
6.2.3 Conductivity against applied fields for colloidal particles and bacteria
The movement of ions and charged particles through solution in response to an
electric field is measured as conductivity [96]. The conductivity of solution with the
same type of ions dissociation and concentration should not change. In this section, the
conductivity of the samples suspended in phosphate pH 7 solutions in corresponding to
the increase in applied field was measured and shown in Figure 6.13. Results showed
that the lower applied field from 20 V to 60 V demonstrated a constant or very slight
increase in conductivity for all the samples but obtained a greater increase of
conductivity after 60 V and lasted untill 120 V. However, the greater applied field
enhanced the attraction field in solution, which increased migration rate of ions and
charged particles and led to a greater electric conduction.
5
7
9
11
13
25
19
Both colloidal particles and bacteria in suspension gave a gradual increase in
conductivity even at lower range of applied field. Figure 6.13 indicates that the lower
conductivity was obtained from TiO2 and liposome. On the other hand, suspension with
dead of S.aureus and E.coli gained better conductivity than live E.coli and S.aureus.
However, total changes in conductivity as applied field increased for all the suspended
materials was marginal, namely less than 6.0 (±0.5) mS cm-1. Results showed that the
total changes in conductivity obtained from colloidal particles and bacteria (live or dead)
were 3.0 (±0.5) mS cm-1and 5.5 (±0.5) mS cm-1 respectively.
15
17
21
23
0 20 40 60 80 100 120 140
Con
duct
ivity
, mSc
m-1
TiO2LiposomeDead SALive SALive E.ColiDead E.Coli
Voltage, V
Figure 6.13 Conductivity of colloidal particles, live and dead E.coli and S.aureus against applied field.
Dead S.aureus and E.coli demonstrated a better conduction in solution and this
might be due to the increase in permeability of surrounding ions through the dead cells.
In contrast, live E.coli and S.aureus which were metabolically active controlled the
penetration of excessive ions, resulting in lower conductivity compared to the dead
cells.
On the other hand, colloidal particles, liposome displayed slight higher
conductivity than TiO2 but lower than biological cells. This is probably because the
colloidal particles are denser than biological cells. Thus, the biological cells responded
slower to an electric field. In addition, TiO2 as a solid particle increases its resistivity to
the surrounding ions when electricity was supplied, thus lower the conductivity.
6.2.4 Electrophoretic mobility of TiO2 and liposome at a range of applied field
Figure 6.14 shows the magnitude of the electrophoretic mobility of both colloidal
particles in a range of applied field. The magnitude of the electrophoretic mobility of
TiO2 and liposome was estimated with Equation 6.14, which the dielectric permittivity
of the medium fixed at 80, f(ka) was 1.5, a fixed value for zeta potential was used in this
assumption and the reduction values of viscosity corresponded to the calculation of
temperature changes as predicted by Equation 6.3.
Figure 6.14. Electrophoretic mobility of TiO2 and liposome for both result from experimental and estimation from calculation.
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
Voltage (V)
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.000 20 40 60 80 100 120 140
Liposome (exp)Liposome (theory)TiO2 (theory)TiO2 (exp)
The magnitude of the electrophoretic mobility of colloidal particles were
predicted and was used to curve-fit with the experimental result for both TiO2 and
liposome. The curve-fitting outcome showed that both the magnitude of the
electrophoretic mobility of colloidal particle fitted closely with theory curve, especially
for TiO2. The result showed that the increase in applied field caused a higher
electrophoretic mobility obtained for TiO2 and liposome. The increase in electrophoretic
mobility of TiO2 and liposome as applied field increased augmented that the attraction
force of the electric field, drew particles and ions towards the electrode. In addition, the
fast-moving ions increase the electrolyte current and elevate the electrolyte temperature.
This reduced electrolyte viscosity, further facilitating the migration of ions and charged
particles to the electrode in a shorter time. Besides, shearing ions could have reduced
the viscous drag of charged particles allowing a faster mobility of particles towards the
electrode.
The electronegativity of TiO2 was greater than liposome as applied field
increased. This might due to the ions from the surrounding moved faster and adhere to
the negatively-charged liposome, hence increased the screens effect and decreased the
magnitude of the electrophoretic mobility. In addition, TiO2 as a solid particle are
denser than semi-permeable particles, this enabled TiO2 to respond greater to
electrostatic force than liposome.
-80
-70
-60
-50
-40
-30
-20
-10
00 20 40 60 80 100 120 140
LiposomeTiO2
Zeta
pot
entia
l (m
V)
Voltage (V)
Figure 6.15. Zeta potential which corresponded to the magnitude of the electrophoretic mobility of TiO2 and liposome.
Figure 6.15 shows zeta potential and corresponding electrophoretic mobility
measured for TiO2 and liposome, where zeta potential of TiO2 indicated an obvious
increased as applied field increased. Meanwhile, zeta potential of liposome
demonstrated a slight increased from 10 V to 80 V but constant at higher applied field.
This is because semi-permeable liposome were less responsive to high applied field.
The ions from solution might penetrate through liposome as electrostatic field increased.
In contrary, zeta potential of TiO2 showed more drastic increase at applied field higher
than 80 V. This showed that TiO2 migrated faster as electrostatic field increased. Beside,
zeta potential for TiO2 was more electronegative compared to liposome, indicating that
the surface charge of TiO2 was more negative than liposome at different applied field,
regardless of size of particles. This is because of the size of liposome is smaller than
TiO2 in this study. Therefore, the influence of particle size on the magnitude of the
electrophoretic mobility was insignificant.
6.2.5 Electrophoretic mobility of live and dead E.coli (TiO as reference sample) 2
As seen in Figure 6.16, the magnitude of the electrophoretic mobility of live and
dead E.coli increased as applied fields increased. Total changes in the magnitude of the
electrophoretic mobility for live and dead E.coli was not much, which recorded for 1.2 x
10-8 m2 -1 -1 -8 V s and 1.5 x 10 m2 -1 V s-1 respectively at the range of applied fields. Besides,
the data indicated that dead E.coli was more negative in the magnitude of the magnitude
of the electrophoretic mobility compared to live E.coli.
Both predicted curves showed a drastic increase in electrophoretic mobility at
higher applied field. This was due to the sudden increase in calculated temperature
change at higher applied field. From the result, the experimental electrophoretic
mobility of live E.coli was closer to the predicted curve but dead E.coli was slightly far
apart from predicted curve. In addition, the predicted electrophoretic mobility of dead
E.coli in applied fields was more negative than the experimental result. This might due
to net negative charges on dead E.coli had been neutralized and decreased from
electrophoretic mobility, hence, demonstrated the deviation from predicted curve.
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
-9.00
-8.00
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.00
1.000 20 40 60 80 100 120 140
Live E.Coli (theory)Live E.Coli (exp)Dead E.Coli (theory)Dead E.Coli (exp)TiO2 (exp)
Voltage (V)
Figure 6.16. Actual and predicted electrophoretic mobility of live and dead E.coli (Electrophoretic mobility of TiO2 as reference).
Figure 6.16 shows that the range of applied field increased the magnitude of the
electrophoretic mobility of either colloidal particles, or live and dead gram-negative
E.coli. This is due to an increase in current which leads to elevation of solution
temperature. The Joule heating process raises the internal energy of ions and particles,
leading to the thickening of the electrical double layer. This causes the charged particles
or cells to move faster to the anode and elicits a higher electrophoretic mobility.
The magnitude of the electrophoretic mobility of live E.coli was more positive
than dead E.coli and TiO2. This might due to the nature live bacteria to resist the change
on surface charges, which live E.coli is able to recover or balance in return the negative
charges on the surface of the cell [102], and ions adhere on its surface would screen the
negative charges, lowering the magnitude of the electrophoretic mobility as the applied
field increase. On the other hand, dead E.coli which was metabolically inactive and was
not able to maintain the amount of charges on surfaces, increased in permeability to ions.
Moreover, the inner lipids might change for dead cell. As a result, negative charges of
dead cells might increase and gave higher electrophoretic mobility.
Live and dead E.coli obtained lower electrophoretic mobility than TiO2. The
results enhanced the assumption that TiO2 as solid particles respond better towards a
strong attraction force of an increase in electric field. This is related to the denser TiO2,
which are impenetrable to ions compared to biological cells.
-40
-35
-30
-25
-20
-15
-10
-5
00 20 40 60 80 100 120 140
Live E.ColiDead E.Coli
Zeta
pot
entia
l (m
V)
Voltage (V)
Figure 6.17. Zeta potential which corresponded to the magnitude of the electrophoretic mobility of live and dead E.coli.
The measured zeta potential calculated from the corresponding electrophoretic
mobility for live and dead E.coli as shown in Figure 6.17. There are increasing in the
electronegativity of zeta potential for live and dead E.coli as applied field increased.
The result demonstrated that live E.coli gave smaller changes in zeta potential as the
electric field increased than of that dead E.coli. This indicated that dead E.coli
possessed higher negative surface charges and responded greater to higher applied field.
6.2.6 Electrophoretic mobility of live and dead S.aureus (TiO2 as reference sample)
Figure 6.18 shows the actual and predicted electrophoretic mobility for live and
dead S.aureus with actual data of TiO2 as a reference for result analysis. The predicted
electrophoretic mobility of the samples showed an increase in negativity of
electrophoretic mobility of suspended materials as applied field increased. However,
experimental results indicated a rather constant magnitude of the electrophoretic
mobility for live and dead S.aureus. Nevertheless, further scrutiny of the results showed
a slight increase in the magnitude of the electrophoretic mobility, which gave -0.43 x
10-8 m2 V-1 -1s and -1.2 x 10-8 m2 V-1 -1s for live and dead S.aureus, respectively,
throughout the range of applied field.
The discrepancy between predicted and experimental result was ascribed to the
thicker peptidoglycan layer of S.aureus, as gram-positive bacteria reduces the effect of
temperature changes on its surface charges caused by an increase in applied field.
Besides, S.aureus has less protein composition and, thus, a less negative surface charge
is obtained. Furthermore, ions from solution would screen the surface charge of
S.aureus. Thus, the effects of enhancing the electric field of solution suspended with
S.aureus did not induce higher magnitude of the electrophoretic mobility for both live
and dead S.aureus. On the other hand, live and dead S.aureus had a less negative
magnitude of the electrophoretic mobility than TiO2. This showed that S.aureus
responded less to the electric field. This may be due to S.aureus is semi-permeable cell
and lesser in density than TiO . 2
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.000 20 40 60 80 100 120 140
Live SA (theory)Live SA (exp)Dead SA (theory)Dead SA (exp)TiO2 (exp)
Voltage, V
Figure 6.18. Experimental and predicted electrophoretic mobility of live and dead S.aureus (Electrophoretic mobility of TiO2 as a reference).
The overall results indicated that dead S.aureus had a slightly higher
electronegative surface charge but not significantly more than live S.aureus. This was
due to thick cell wall peptidoglycan layer which increased the resistivity of ions
penetrating both live and dead S.aureus. Although dead S.aureus had no metabolic
activity, the thick layer of cell wall was able to prevent intracellular ions from being lost
to the electrolyte and to reduce the excessive permeability of extracellular ions passing
through dead cells. Consequently, the magnitude of the electrophoretic mobility for both
live and dead S.aureus had not much different.
Figure 6.19. Zeta potential which corresponded to the magnitude of the electrophoretic mobility of live and dead S.aureus.
Zeta
pot
entia
l (
mV
) Voltage (V)
-40
-35
-30
-25
-20
-15
-10
-5
00 20 40 60 80 100 120 140
Live SADead SA
The result for zeta potential measured for live and dead S.aureus was in agreement
with its electrophoretic mobility. The difference between live and dead strain was less
obvious as depicted in Figure 6.19. However, the results showed an increase in zeta
potential at higher applied field, particularly, after 80 V. This implied that the ions in the
diffuse layer moved faster and more freely at higher applied field. This was because the
current of solution increased and elevated temperature changes as the applied field
increased. The increase in temperature reduced solution viscosity as ions and cells
gained more kinetic energy to migrate faster as an electric field was applied.
Consequently, a greater zeta potential was measured at higher applied field.
6.2.7 Electrophoretic mobility of live E.coli and S.aureus
A comparison of the magnitude of the electrophoretic mobility and zeta potential
of live strains of E.coli and S.aureus is shown in Figure 6.19. The result indicated that
an increase in applied field rose in the magnitude of the electrophoretic mobility and
zeta potential. However, the increase of live E.coli and S.aureus was of lower
magnitude than TiO2. This was due to the denser particle, TiO2 responded greater to
higher applied field compare to semi-fluid particles, such as live cells. Besides, the data
also demonstrated a lower negative surface charge for live E.coli compared to S.aureus.
The surface charge of E.coli was screened by the surrounding ions as its cell wall is
thinner than S.aureus, thus E.coli enable the faster exchange of ions in and out of the
cell.
Elec
troph
oret
ic m
obili
ty, m
2 V-1
s-1 (x
10
-8)
-2.50
-2.00
-1.50
-1.00
-0.50
0.000 20 40 60 80 100 120 140
-30
-25
-20
-15
-10
-5
0
Live SA (EPM) Live E.Coli (EPM)Live SA (Zeta potential)Live E.Coli (Zeta potential)
Zeta potential (mV
)
Voltage, V
Figure 6.20. Electrophoretic mobility and the corresponded zeta potential of live E.coli and S.aureus.
Live S.aureus recorded more stable and consistent zeta potential and
electrophoretic mobility across the range of applied field. This was because the thicker
cell wall peptidoglycan layer of these gram-positive bacteria reduced the mobility ions
across the cells. Hence, the net surface charge was less influenced by applied field. In
conclusion, live E.coli was more responsive to the effects of applied fields compare to
S.aureus. A greater applied field induced a greater increase in electrophoretic mobility,
as current increased and elevated solution temperature increased. However, live bacteria
might die if temperature is too high, mimicking death by burning.
6.3 Effects on different time interval between electrophoretic mobility measurements of live E.coli and S.aureus
The magnitude of the electrophoretic mobility of the cells was assumed to be
constant in an unchanged condition. Surface charges of live cells in unchanged
condition in different time intervals between measurements were studied as calibration
to the changes of surface charges. The study was based on the concept of resting
potential membrane. For instance, a voltmeter is attached to the two terminals of a
battery and voltage difference will be measured across the two terminals. Likewise, if a
voltmeter is used to measure voltage across the cell wall (inside versus outside) of a
cardiomyocyte or live cell, it will be found that the inside of the cell has a negative
voltage (measured in millivolts; mV) with respect to the outside of the cell (which is
referenced as 0 mV). In resting conditions, this is called the resting membrane potential.
The negative voltage inside the cell (negative membrane potential) may transiently
become positive owing to the generation of an action potential whenever an appropriate
stimulation is given. Membrane potentials result from a separation of positive and
negative charges (ions) across the membrane, similar to the plates within a battery that
separate positive and negative charges [150, 151]. Cell walls are typically permeable to
only a subset of ionic species. These species usually include potassium ions, chloride
ions, bicarbonate ions, and others. Thus, electric attraction on the bacteria might initiate
change or shock on the live bacteria which may lead to the changes of the surface
charges.
Two different set of time interval were investigated which one set was less than
10s (1s, 3s, 5s and 7s) and another set of time interval was longer than 10s (10s, 60s,
80s and 120s).
Figure 6.21 (a)-(d) demonstrates the result for measurement of different time
interval for less than 10s between measurements. Both dotted lines were the average
zeta potential for live E.coli and S.aureus respectively. If the magnitude of the
electrophoretic mobility of bacteria was unchanged, results along the time interval
would remain at the mean position. From the result, both live E.coli and S.aureus
recorded erratic results for time interval less than 10 s. Result for time interval 1 s
obtained the most constant zeta potential as the time interval between measurements
were too short for the change of cell surface charges.
Figure 6.21 (a). Zeta potential of live E.coli and S.aureus for time interval at 1s.
Zeta
pot
entia
l, m
V
Rest time duration, s
-30
-25
-20
-15
-10
-5
0
50 1 2 3 4 5 6 7 8 9 10 11
Live E.Coli
Live S.aureus
Rest time duration, s
Zeta
pote
ntia
l,m
V
-30
-25
-20
-15
-10
-5
0
50 3 6 9 12 15 18 21 24 27 30 33
Live E.Coli
Live S.aureus
Figure 6.21 (b). Zeta potential of live E.coli and S.aureus for time interval at 3s.
Rest time duration, s
-30
-25
-20
-15
-10
-5
0
5 0 5 10 15 20 25 30 35 40 45 50 55
Live E.Coli
Live S.aureus
Zeta
pot
entia
l, m
V
Figure 6.21 (c). Zeta potential of live E.coli and S.aureus for time interval at 5s.
Rest time duration, s
-30
-25
-20
-15
-10
-5
0
50 7 14 21 28 35 42 49 56 63 70 77
Live E.Coli
Live S.aureus
Zeta
pot
entia
l, m
V
Figure 6.21 (d). Zeta potential of live E.coli and S.aureus for time interval at 7s.
However, the magnitude of the electrophoretic mobility gave more erratic results as
time intervals increased and can be observed from Figure 6.21 (c) to (d). This might be
due to the screens effect of bacteria and recovering of negative surface charges of
bacteria that result the inconsistent zeta potential as time interval increased. Besides,
when the supplied voltage cut off after one measurement, the moving particles stopped
but still carried a momentum which would keep them swirling and vibrating in the
solution for less 10s. This motion is considered as a retardant force which could have
reduced the fast response of particles to the electric field when the next measurement
starts. The swirl force of the particles appeared to be stronger than the net charge on the
surface charge of particles which carried them to the electrode. Thus a lower reading of
zeta potential at shorter time interval which was less than 10s obtained for live E.coli
and S.aureus.
Zeta potentials of live E.coli and S.aureus at different time interval between
measurements for 10s, 60s, 80s and 120s are depicted in Figure 6.22 (a)-(d). Live E.coli
showed constant results for all different time interval. Meanwhile, live S.aureus
obtained stable zeta potential at time interval of 10s and 60s but more erratic at time
interval of 80s and 120s.
Rest time duration, s
-70
-60
-50
-40
-30
-20
-10
08 18 28 38 48 58 68 78 88 98 108
Zeta
pot
entia
l, m
V
Live E.Coli
Live S.aureus
Figure 6.22 (a). Zeta potential of live E.coli and S.aureus at each rest time of 10 s.
Zeta
pot
entia
l, m
V
Rest time duration, s
-70
-60
-50
-40
-30
-20
-10
020 120 220 320 420 520 620 720
Live E.Coli
Live S.aureus
Figure 6.22 (b). Zeta potential of live E.coli and S.aureus at each rest time of 60 s.
Zeta
pot
entia
l, m
V
Figure 6.22 (c). Zeta potential of live E.coli and S.aureus at each rest time of 80 s.
Rest time duration, s
-70
-60
-50
-40
-30
-20
-10
060 160 260 360 460 560 660 760 860
Live E.Coli
Live S.aureus
-70
-60
-50
-40
-30
-20
-10
0110 230 350 470 590 710 830 950 1070 1190 1310
Live E.Coli
Live S.aureus
Zeta
pote
ntia
l,m
V
Rest time duration, s
Figure 6.22 (d). Zeta potential of live E.coli and S.aureus at each rest time of 120 s.
Zeta potential of live E.coli and S.aureus at time interval for more than 10s was
higher than zeta potential for time interval less than 10 s. Zeta potential for live E.coli
and S.aureus at time interval less than 10s were -7 mV and -12 mV respectively.
Meanwhile, zeta potential for time interval more than 10s for live E.coli and S.aureus
were -16 mV and -31 mV. The different was due to the live cells needed a longer time
interval to restabilize charges on cell surface after initiated by electric field.
Consequently, the bacteria would suspend more stable and recover the amount of
surface charge at time interval for more than 10s between measurements.
6.4 Summary on the effects of different temperatures, applied field and time interval on surface charge
Results indicated that Helmholtz-Smoluchowski predicted curve fitted better
with experimental result of the magnitude of the electrophoretic mobility of colloidal
particle compared to bacteria. Besides, the negativity of the magnitude of the
electrophoretic mobility for liposome appeared to be greater than that of TiO2, this is
due to a lower amount of heat absorption is sufficient to increase the kinetic energy of
particles to migrate towards the electrode. In different temperature, the magnitude of the
electrophoretic mobility of dead E.coli was erractic and greater than live E.coli. The
result of the magnitude of the electrophoretic mobility of live E.coli displayed mild
increase at various temperatures that demonstrated live E.coli was viable at this range of
temperature. On the other hand, result indicated only a slight increased in negative
magnitude of the electrophoretic mobility of live S.aureus as the temperature increased.
This showed that live S.aureus was not affected much by this range of temperature. At
higher temperature, dead S.aureus obtained higher electronegative than live S.aureus
due to live S.aureus are metabolically active and able to resist the change on its surface
143
charge. The magnitude of the electrophoretic mobility for live S.aureus at different
temperature was greater than live E.coli. This might due to the thinner cell wall of E.coli
was easier to be screened by the ions from solution, decreased the net negative charges
on surface.
The increase in applied field increased the magnitude of the electrophoretic
mobility of bacteria and colloidal particles. The applied field increased the current and
elevated temperature of electrolyte. Thus, this reduced viscosity of electrolyte, gave a
higher magnitude of the electrophoretic mobility. In addition, increased in higher
applied field increased the attraction force on charged particles or cells. The comparison
result of the magnitude of the electrophoretic mobility showed that TiO2 possessed
greater net negative charge than liposome in the range of applied field. This is due to the
proportional relationship between density of suspended material and applied force. This
explained the lower the magnitude of the electrophoretic mobility of liposome
compared to TiO2. Besides, results showed live E.coli obtained smaller changes in
electrophoretic mobility as applied field increased and comparatively dead E.coli was
more negative in electrophoretic mobility. For gram-negative bacteria, result indicated
dead S.aureus obtained a slightly higher electronegative surface charge but not
significant compared to live S.aureus. This was due to the thicker peptidoglycan layer of
cell wall in S.aureus increased the resistivity of the ions to penetrate through the cells
either for both live and dead S.aureus. Although dead S.aureus has no metabolic activity
but the thick layer of cell wall was able to avoid the inner ions lost to solution and
reduced the excessive permeability of external ions to pass through the dead cells.
Consequently, the magnitude of the electrophoretic mobility for both live and dead
S.aureus has not much discrepancy. Besides, a lower negative surface charge for live
E.coli compare to S.aureus at different applied field. This might be because of the
144
surface charge of E.coli has been screened by the surrounding ions as the cell wall is
thinner than S.aureus which has enable the faster exchange of ions inner cell to
surrounding and vice versa.
Different time interval between measurements for the unchanged conditions
indicated that zeta potential of live E.coli and S.aureus was able to restabilize the
surface charge for time interval of more than 10s. Hence, higher zeta potential was
measured for time interval more than 10s between measurements.
145
CHAPTER 7
Polarization of Bacteria and Colloidal Particles
7.0 Polarization of bacteria and colloidal particles
Bacteria which carry negative charges in phosphate buffer solution pH 7 attract
oppositely-charged ions to adhere to the cell surface. These opposing charges on cells
form dielectric properties in bacteria. Bacteria charges on the cell surfaces tend to
polarize when an electric field is applied. Low frequency dispersion gives rise to an
effective dielectric constant at low frequency (<1000 Hz). The cell wall of a bacterial
cell is electrically similar to an ion exchange resin [152].
The polarization, P induces the cell dielectric is also proportional to the applied
electric field vector E.
EP oeεχ= (7.1)
where εo is the dielectric permittivity of bulk solution with a of 80, , χe is the dielectric
susceptibility. The dielectric characteristic of cells and colloidal particles are identical
when using a parallel plate capacitor. Therefore, the polarization is very much related to
the dielectric displacement vector D, denotes the total surface charge density induced in
the dielectric, as given by the formula in (7.2).
PED o += ε (7.2)
Substituting the term for P in Equation 7.2 gives rise to Equation 7.3.
146
( ) EEED oeoeo εχεχε +=+= 1 (7.3)
If Q is the total charge on cell surface and A is the area of disposable cells, then D=Q/A.
Besides, E=V/d, where V is the potential difference across cells and d is the thickness of
samples. Substituting the relationships for D and E in Equation 7.3, the following
Equation is obtained.
( )dV
AQ
oe εχ+= 1 (7.4)
The capacitance, C of the cell is defined as the ratio of the charge on either plate to the
potential difference between the plates, C=Q/V [153]. Hence, the relationship between
the capacitance, C and dielectric susceptibility, χe is expressed as
( eoCVQC χ+== 1 ) (7.5)
where dAC oo /ε= .In this study, εo was 80, area, A was 1.26 × 10-5 cm2 and d was 8.8
cm. Meanwhile, cell capacitance was obtained from the phase different of measurement.
GX c
/1tan 1−
=θ (7.6)
CXC ω/1−= (7.7)
θ is the phase different, Xc is capacitive reactance, G denotes the conductivity of the
solution. ω is defined as angular speed, 2πf, and depends on the frequency of the
147
alternating current supply. Hence, the polarization of cells and particles, can be
calculated from the above formula derivation.
7.1 Clausius-Mossotti Approximation
Clausius-Mossotti approximation is used to describe the effective conductivity or
susceptibility of mixtures and materials that containing several phases, and this model
was introduced by Ottavanio Fabrizio Mossotti in 1846 [154]. The Clausius-Mossotti
factor, K(ω) is shown in Equation 7.8.
( ) **
**
2 mp
mp
εεεε
ω+
−=Κ (7.8)
ωσ
εε ppp i−=*
ωσ
εε mmm i−=* and where, are complex permittivities of the particles
and the medium respectively, with σ is conductivity, ε is denoted as permittivity, ω
represents angular frequency of the applied electric field and 1−=i .
Clausius-Mossotti factor, K(ω), which is frequency dependence indicates that the
force acting on the charged particles varies with frequency. This also relies on the
relative polarizability of the charged particles with respect to the surrounding medium.
The charged particles is induced to move towards a region where the electric field
gradients are the strongest (K(ω)>0, positive dielectricphoresis) or towards a region
where the electric field gradients are the weakest (K(ω)<0, negative dielectricphoresis).
In electrokinetic manipulation, the real part of the Clausius-Mossotti factor is a
determining factor for the dielectrophoretic force on a particle, where as the imaginary
part is a determining factor for the electrorotational torque on the particle. Other factors
are describing the geometries of the particle to be manipulated and the electric field.
148
In dielectrophoresis manipulation, the electrical field is mainly dropped across
the outmost membranes of the cells at low frequencies, causing the cells behave as a
poor conductive spheres [155]. As frequency increases, the applied field gradually
penetrates into the cells. This enhances the conductivity of the cells with high
permittivity of the cell interior [155]. At low frequency, fCM ( εσω << ) [156],
Equation 7.8.1 can be estimated. On the other hand, high frequency, fCM ( εσω >> )
[156] is shown in Equation 7.8.2.
( ) ( )mpmpCMf σσσσ 2/ +−= (7.8.1)
( ) ( )mpmpCMf εεεε 2/ +−= (7.8.2)
Consequently, there is possibility of cells exhibit negative dielectrophoresis at
low frequency if σp <σm, (K(ω)> 0 ) and positive dielectrophoresis at high frequency
if ε > εp m (K(ω) < 0 ) [157].
Most of the biological cells behave as dielectrically polarized particles in a
non-uniform electric field. Therefore, the characteristic of cell can be manipulated by
dielectrophoresis by separating different types of cells based on the differences in the
dielectrical polarizabilities among the cells [158]. Different frequencies were
determined to estimate the cells permittivities, εp were estimated with Equation 7.8.1
and 7.8.2.
149
7.2 Result and discussion on polarization of live cells and colloidal particles
The frequency of the alternating current was set at 10 Hz. The polarization of each
sample is shown in Table 7.1. Results indicated that the polarizability of live and dead
E.coli was greatest. Live cells showed greater polarizability than dead cells, as
demonstrated by both E.coli and S.aureus. Dead cells recorded half reduction in
polarizability compared to live cells. On the other hand, polarizability of TiO2 and
liposome in phosphate buffer pH 7 were lower than the bacteria cells.
The polarization of cells relies on the permeability of particles to the ions in the
solutions. The high polarizability of E.coli indicated that the shearing of ions around
cells was greater, causing more cellular rotation. This phenomenon might the reason of
the low magnitude of the electrophoretic mobility obtained. Besides, Table 7.1 showa
that the dead cells exhibited a lower polarization than live cells, demonstrating that the
surface charge of dead cells was screened by surrounding ions. The loss in metabolic
activity prevented dead cells from recovering their surface charge. On the other hand,
colloidal particles, TiO2 and liposome obtained a low polarizability compared to
Samples Phase
Different θ,
(radian)
Capacitance
C,(Farad)
Polarization
(μC/cm2)
TiO2 0.500 0.0414 0.0903
Liposome 0.662 0.0225 0.0195
Live S.aureus 0.360 0.1247 1.1403
Dead S.aureus 0.540 0.0783 0.6674
Live E.coli 0.269 0.1703 2.2064
Dead E.coli 0.354 0.1007 1.2262
Table 7.1. Polarization of bacteria and colloidal particles.
150
bacteria cells. This may be atributed to the particles are low in permeability to ions from
solutions.
7.3 Result and discussion on low frequency dependence Clausius-Mossotti formula
The forces act on charged particles depend on the frequency of the electric apply
field. This can be estimated with Clausius-Mossotti formula, K(ω), which considers the
relative polarizability of the charged particles with respect to the suspending electrolyte
[159]. At low frequency, the change of conductivity cells was predicted with Equation
7.8.1, which the range of frequency is less than 1.0 Hz and shows in Figure 7.1.
The result shows conductivity of liposome, live E.coli and S.aureus increased as
frequency increased. After frequency 0.8 Hz, increased in conductivity cells were
drastic. This demonstrated that higher applied frequency increased the conductivity of
cells. Result of conductivity increased for liposome was lower than live E.coli and
S.aureus, this was because of the lower electrolyte conductivity suspends with liposome,
that obtained from measurement.
At higher frequencies, Equation 7.8.2 was used to predict the change of permittivity
particles and ratio of ε / εp m. The electrolyte permittivity, εm was fixed at 80 for the
result calculated, thus only one predicted result as shown in Figure 7.2. Result
demonstrated a sharp increased in particle’s permittivity below 200 Hz. This indicated
that a more stable result can be obtained from 200 Hz until 1000 Hz, which the results
shows that the permittivities were unchanged. The ratio of ε / εp m demonstrated the same
result as the increased in particle permittivity. The negative sign in predicted results of
particle permittivity can be ignored as there is no direct relation between optical
electronegativity and electronic polarizability [160].
151
Con
duct
ivity
of
cel
ls, σ
p , S
/m
0
5
10
15
20
25
30
35
40
45
50
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
LiposomeLive E.coliLive S.aureus
Frequency, Hz
Figure 7.1. Predicted conductivity of liposome, live E.coli and S.aureus with frequency less than 1.0 Hz.
Ratio of ε
p /εm
Parti
cle
perm
ittiv
ity, ε
p
-190
-185
-180
-175
-170
-165
-160
-1550 200 400 600 800 1000 1200
-2.35
-2.30
-2.25
-2.20
-2.15
-2.10
-2.05
-2.00
-1.95
Particle permittivityratio of Ep/Em
Frequency, Hz
Figure 7.2. Particle permittivity and compare to the ratio of particle permittivity over the electrolyte permittivity were estimated in higher frequency
152
7.4 Summary of polarizability of bacteria and colloidal particle
The live bacteria recorded a higher polarizability compare to colloidal particles.
This was because of semipermeable cell membrane of live cells hasten the ions
exchange between the cells and external environment. The highest polarization was
obtained from E.coli, which E.coli possessed thinner cell wall peptidoglycan layer
compared to S.aureus. The conductivity and permittivity of cells increased as the
frequency increased. However, the permittivity of cells were shown constant at higher
frequency (>200Hz).
153
CHAPTER 8
Conclusion and Future Work
8.0 Discussion and conclusion
Studies were carried out on the surface charge of samples at different
temperatures, applied fields, under different chemical conditions and the polarizability
of bacteria were investigated. The negative surface charge of S.aureus yielded higher
magnitude of the electrophoretic mobility than E.coli under all study conditions. The
major difference between these two bacteria is the thickeness of peptidoglycan layer,
with that of gram-positive S.aureus being greater than gram-negative E.coli. Among the
samples, colloidal particles, TiO2 recorded the greatest negative magnitude of the
electrophoretic mobility compared to bacteria. This was because solid particles had no
metabolic activity and comprised of a constant surface charge.
It was shown that bacteria showed an increase in the magnitude of the
electrophoretic mobility while number of washes increased. Different chemical
environments had affected the magnitude of the electrophoretic mobility of samples.
The result showed that E.coli obtained the lowest magnitude of the electrophoretic
mobility compared to S.aureus in different chemical environments. At various pH, dead
cells of both E.coli and S.aureus obtained lower the magnitude of the electrophoretic
mobility than live cells. This is mainly because of the dead cells had no metabolic
activity to recover their surface charges and most of the charges screened by
surrounding ions. An increase in pH increased the magnitude of the electrophoretic
mobility of bacteria and TiO2 which is due to the higher concentration of hydroxide
groups that increased the net negatively-charged of samples. On the other hand, an
154
increasing on ionic strength of NH4Cl reduced the magnitude of the electrophoretic
mobility of samples. This is because of the share current carried by the charge particles
and cells decreased as the free ions in solution increased. However, bacteria suspended
in an increased of NaCl ionic strength obtained opposite results. This might due to the
excessive influx of Na+ ions and increased the anionic lipid of cells. Thus, higher ionic
strength increased the net negative charges on bacteria and higher electrophoretic
mobility obtained. The magnitude of the electrophoretic mobility of live bacteria in
NaCl solution was compared with the numerical approximation of the Donnan potential.
Results indicated that both approaches fitted well at greater ionic strengths of NaCl
solution.
Results indicated that the Helmholtz-Smoluchowski predicted curve fitted better
with experimental results of the magnitude of the electrophoretic mobility of colloidal
particles at a range of temperatures. The increase in temperature increased the
magnitude of the electrophoretic mobility of suspended materials. This is because
viscosity of solution decreases and hastens the electrophoretic mobility. Both the
magnitude of the electrophoretic mobility of live E.coli and S.aureus displayed mild
increases at various temperatures, showing that they resist to the changes of temperature
on its surface charges. At higher temperature, dead S.aureus displayed higher
electronegativity than live S.aureus because the latter is metabolically active and able to
regain their initial surface charge. The magnitude of the electrophoretic mobility of
E.coli at different temperatures was greater than live S.aureus. The thinner cell wall of
E.coli was more easily screened by the ions in solution. The higher the electrophoretic
mobility of S.aureus in overall testing might due to the smaller size of S.aureus
compared to E.coli.
155
The increase in applied field increased the magnitude of the electrophoretic
mobility of bacteria and colloidal particles. The applied field increased the current and
elevated solution temperature. Subsequently, this reduced the viscosity of solution.
Besides, comparison of the magnitude of the electrophoretic mobility showed that TiO2
was more negative in magnitude of the electrophoretic mobility than liposomes in the
range of applied field. This is due to the proportional relationship between density and
applied force. For biological cells, live E.coli displayed smaller changes in the
magnitude of the electrophoretic mobility as applied field increased and, comparatively,
dead E.coli was more negative in the magnitude of the electrophoretic mobility than live
E.coli. This might due to increase in permeability of dead cells reduce the negative
surface charge of dead E.coli. For gram-negative bacteria, the magnitude of the
electrophoretic mobility of both live and dead S.aureus did not differ much. This was
because the thicker cell wall peptidoglycan layer of S.aureus increased the resistivity to
ions penetrating both live and dead S.aureus. Although dead S.aureus has no metabolic
activity, its thick cell wall was able to prevent cellular ions from being lost to the
electrolyte and reduced the excessive influx of external ions. The data demonstrated a
lower negative surface charge for live E.coli compared to S.aureus. The surface charge
of E.coli had been screened by the surrounding ions as its cell wall was thinner than
S.aureus, enabling a quicker exchange of ions between the cell interior and exterior.
Different time intervals between measurements for the same condition indicated
that the zeta potential of live E.coli was able to restabilize the ions for duration of more
than 10 s after the first initiation of an electric field compared to live S.aureus which
possesses a thicker cell wall layer peptidoglycan. This was because the thicker cell wall
of S.aureus reduced the rate ion exchange between the cell interior and exterior, causing
the surface charge of S.aureus to be less stable compared to live E.coli for the same time
156
interval. Fluctuations in the electrophoretic mobility of S.aureus at longer time intervals
such as 60 s and 120 s were due to screen effects of cells in electrolyte. On the other
hand, live bacteria recorded a greater polarizability compared to colloidal particles. This
is because the thin cell membrane of live cells enhances ions exchange between the cell
interior and exterior. The greater polarization of E.coli over S.aureus is due to the
thinner cell wall peptidoglycan layer of the former.
8.1 Future Works
There are a few research suggestions on the study of bacterial surface charge can
be continued. The different ionic strengths of the chemical conditions employed in this
study can be further extended. Besides, effects of temperature on the surface charge of
bacteria can be studied at temperatures lower than room temperature. For instance,
freezing conditions might bring about a change in bacterial surface charge to enhance
survival. A detailed study of surface charge at such conditions can improve existing
freezing methodology employed to kill bacteria.
Another future investigation that can be carried out is to load different types of
drugs in to bacteria and measure electrophoretic mobility in order to gain more insight
into bacterial response to drugs. Such a study can further improve on current usage of
drugs to control bacterial growth or to alter bacterial surface charge.
The proteins of different strains of live bacteria can be extracted and their
electrophoretic mobility recorded bacterial proteins might be useful to that disease.
The study of live bacterial surface charge and shorter time interval between
measurements can be further extended to get clearer picture of how changes in surface
charge may be manipulated in pursuit of a cure for memory loss in humans. The spread
157
of cancer cell in the body are due to the imbalance or abnormality of ions affecting the
surface charge of normal cells. Thus, more research can be carried out find a better cure
for cancer.
The properties of surface charges of different blood diseases can be collected. This
provides information on the surface charge to indentify an accurate treatment for the
diseases. This is because the fundamental study of surface charge of unicellular
organisms is essential in developing various fields such as industry applications,
medicine and food.
158
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LIST OF PUBLICATIONS
a) International Publications
a1. P.F.Lee , W.A.T.Wan Abdullaha and Misni Misranb , “Surface Charge Comparison of Liposome and TiO2”, An Indian Journal :Biochemistry, Trade Science Inc., BCAIJ, 2(1), pg 18-23, 2008.
a2. P.F.Lee , W.A.T.Wan Abdullaha and Misni Misranb ,
“Electrophoretic Mobility of Different Sizes Synthesized Titanium Dioxide (TiO2) based Microemulsion”, Nano Science and Nano technology, An Indian Journal, accepted 18th February 2009.
c) Proceedings
1. Title of paper : Effects on Electrophoretic Mobility of different R values Microemulsion to form Titanium Dioxide (TiO ) 2
a Authors : P.F.Lee , W.A.T.Wan Abdullaha and Misni Misranb
Date : 23-25 August, 2007 Activity : 12th Asian Chemical Congress (12ACC) Venue : PWTC Role : Poster presentation, PTC 078, Abstract pg 153.
2. Title of paper : Comparison of the Surface Charge between Liposome and TiO2
a
Authors : P.F.Lee , Misni Misranb a and W.A.T.Wan AbdullahDate : 26-28 December 2007 Activity : National Physics Conference Venue : Terengganu Heritage Bay Club, Pulau Duyung Role : Oral presentation, O79, Abstract pg 64
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