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UTILIZATION OF TRANSIENT ELECTROPORATION IN
INTENSIFIED BIOPROCESSING: A STUDY FOR THE
ENHANCEMENT OF L-GLUTAMATE PRODUCTION BY
CORYNEBACTERIA
A thesis submitted to the Heriot Watt University for the degree of
DOCTOR OF PHILOSOPHY
by
Mostafa Zahid Sharif
Department of Chemical Engineering
School of Engineering and Physical Sciences
Heriot Watt University
Edinburgh EH14 4AS
August, 2008
This copy of the thesis has been supplied on condition that anyone who consults it is
understood to recognise that the copyright rests with its author and that no quotation
from the thesis and no information derived from it may be published without the prior
written consent of the author or the University (as may be appropriate)
Abstract
Although L-glutamate production by Corynebacteria fermentation has been wellestablished in the biotechnology industry since 1960’s, the demand for this building
block for human consumption is increasing enormously. Researchers have therefore been
trying to improve the yield and productivity of amino acids by developing genetically
modified organisms or through the use of metabolic engineering tools. In this study, a
new approach by which the utilization of transient electropermeabilization for increasing
the membrane permeability of these bacteria to L-glutamate secretion will be
investigated, thus potentially enhancing the yield of glutamic acid production.
Micrococcus glutamicus (a strain of Corynebacteria) cultivated in CGXII
Minimal Medium was successfully used to produce L-glutamate under different growth
conditions i.e., biotin limitation, surfactant (Tween 40) addition and ethambutol addition.
This study clearly showed that the production of L-glutamate is influenced by the
concentration of these agents and by their time of addition into the fermentation medium.
The highest amount of L-glutamate was found to be 57mM at a biotin concentration of
1µg l-1. A simple method based on centrifugation and acid-base addition was developed
in which L-glutamate could be separated with a purity of 90% from the fermentation
broth. This research confirmed that cell growth, viability, substrate consumption and Lglutamate production by M. glutamicus are notably affected by the increase of medium
osmolality (caused by the addition of NaCl). This phenomenon is often observed because
of the use of high concentration industrial medium or accumulating amino acids into the
extracellular medium during fermentation.
This study confirmed that the conductivity of medium in which cells are
suspended represents a major barrier for excreting the intracellular protein or enzyme by
membrane electropermeabilization, regardless of the electric field strength and the
number of pulses applied. The electroenhancement of L-glutamate secretion by the
above-mentioned treatments is limited due to the high conductivity of fermentation broth.
However, an increase (about 19%) of total protein excreted from M. glutamicus as
compared to the control was obtained in cells suspended in a less conductive medium
(dH2O) and electroporated at 12.5kV cm-1, 200 and 25µF with 3 pulses. It was also
observed that electroporation factors, especially field strengths, pulse numbers, medium
conductivity and the configuration of cells have a major impact on cell viability. The
yield of electrosecretion will be limited due to the reduction in cell viability during
continuous microbial fermentation and metabolite extraction by the high intensity electric
pulses used. This research has established many of the factors requiring consideration
when using electroporation for bioprocess intensification.
ii
Dedication
To my family, for getting me this far
iii
Acknowledgements
First of all I would like to thank my supervisors Dr. Derek Wilkinson and Dr.
Mark T. Bustard for their continuous support and guidance during my research at the
Department of Chemical Engineering, Heriot Watt University. Without their help and
encouragement, this study would not have been completed. I would like to give my
special appreciation to Dr. Bustard for setting up the frames of this study, for his efforts
to explain the research clearly, for his continued encouragement to work coherently, and
for good scientific discussions and productive advices. He offered direction, penetrated
criticism and was always there when I needed him.
An exceptional gratitude goes to Dr Nicholas Willoughby, who has provided me
the valuable supports, good suggestions, and logical arguments during the thesis-writing
period. I will always be grateful for everything he did for me. I wish to express my
cordial appreciation to Eileen McEvoy and Marian Miller for their assistance and
counseling throughout this research. Special thanks to Dr Wilf J. Mitchell, Biological
Sciences for allowing me to use the French Press. Many thanks to Cameron Smith and
Ann Blyth for their great help whenever it was needed.
I would like to thank my colleagues, Theodora Tryfona and Neil Hollow for their
supports and insights. I am grateful to all my friends at Heriot Watt University, in
particular Nadimul Faisal, Kumar Patchigolla and Carla Buonacucina for being the
surrogate family during the time I stayed here. I am also obliged to my friends in
Edinburgh, specially Polok, Chapal, Selim, Mannu, Shuvra, Gora and Shahed for giving
me the pleasure. Special thanks to my friends (Sidhartha, Aunik, Robi, Niladri and
Subroto) in Bangladesh who physically might have been many miles away, but mentally
were with me all the time.
Finally, I am forever indebted to my parents, brother (Bablu), sister (Tonni) and
uncles for their understanding, endless patience, and encouragement when it was most
required.
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Table of Contents
Section
Title
Page
Abstract
ii
Dedication
iii
Acknowledgements
iv
Table of Contents
v
List of Figures
x
List of Tables
xvi
1.
Chapter ONE: Introduction
1
2.
Chapter TWO: Literature Review
9
2.1.
Corynebacteria
9
2.1.1.
Taxonomy and cell wall of Corynebacteria
9
2.1.2.
Central metabolism of Corynebacteria (sugar uptake, glycolysis
and TCA cycle)
13
2.1.3.
Anaplerotic pathways of Corynebacteria
18
2.1.4.
Uptake and assimilation of ammonium in Corynebacteria
21
2.1.5.
Metabolic engineering and metabolic flux analysis (MFA) of
Corynebacteria
26
2.2.
Industrial production of amino acids
31
2.2.1.
Introduction
31
2.2.2.
Fermentative production of amino acids by Corynebacteria
34
2.2.3.
Factors affecting during amino acids production in Corynebacteria
37
2.2.4.
Mechanisms of L-glutamate efflux in Corynebacteria
39
2.3.
Electroporation
43
2.3.1.
Introduction
43
2.3.2.
Theory and kinetic studies of electropermeabilization
46
v
2.3.3.
Factors affecting cell electropermeabilization
51
2.3.3.1. Cellular factors
52
2.3.3.2. Physiochemical factors
53
2.3.3.3. Electrical parameters
58
3.
Chapter THREE: Production of L-glutamate by
Fermentative cultivation of Corynebacteria under
Different Growth Conditions
61
3.1.
Introduction
61
3.2.
Material and Methods
67
3.2.1.
Chemicals
67
3.2.2.
Organism and cultivation
67
3.2.3.
Microbial growth measurement
68
3.2.4.
Calculation of maximum specific growth rate (µmax)
69
3.2.5.
L-glutamate production in M. glutamicus under biotin-limited
condition
69
3.2.6.
L-glutamate production in M. glutamicus by surfactant addition
72
3.2.7.
L-glutamate production in M. glutamicus by ethambutol addition
73
3.2.8.
Quantification of glucose, L-glutamate and other amino acids
73
3.2.9.
Recovery of glutamic acid from fermentation broth
76
3.3.
Results
78
3.3.1.
Growth properties of the different strains of Corynebacteria in a
defined medium
3.3.2.
78
L-glutamate production by the different strains of Corynebacteria
under biotin-limited condition
3.3.3.
80
Secretion of L-glutamate produced by M. glutamicus due to
surfactant addition
3.3.4.
3.4.
85
Secretion of L-glutamate produced by M. glutamicus due to
ethambutol addition
90
Discussion
94
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4.
Chapter FOUR: Enhancement of the Secretion of
L-glutamate Produced under Biotin Limited Fermentation
of M. glutamicus by Electropermeabilization
105
4.1.
Introduction
105
4.2.
Material and Methods
112
4.2.1.
Chemicals
112
4.2.2.
Organism and cultivation
112
4.2.3.
L-glutamate production in M. glutamicus under biotin limited
condition
112
4.2.4.
Electroporator and pulse treatment
112
4.2.5.
Quantification of glucose, L-glutamate and other amino acids
113
4.2.6.
Preparation of crude extract
113
4.2.7.
Determination of total protein, GDH and MDH
115
4.3.
Results
123
4.3.1.
Effect of electric pulses on the secretion of L-glutamate produced in
M. glutamicus
4.3.2.
123
Effect of providing resting time and controlling the temperature of
samples between two pulses while pulsing repetitively on the secretion
of L-glutamate produced in M. glutamicus
4.3.3.
125
Effect of electric field strengths, providing resting time and controlling
the temperature of samples while pulsing repetitively on the release
of total protein and enzymes (GDH and MDH) by M. glutamicus
4.3.4.
127
Effect of the type of strains, providing resting time and controlling the
temperature of samples while pulsing repetitively on the release of
total protein and enzymes (GDH and MDH) by E. coli
4.3.5.
Effect of the pre-treatment of cell permeabilizing agent (DTT) before
pulsation on the release of total protein and enzymes (GDH and MDH)
4.3.6.
4.4.
129
131
Effect of the pulsing media in which cells are suspended on the release
of total protein and enzymes (GDH and MDH) by M. glutamicus
132
Discussion
135
vii
5.
Chapter FIVE: Transient Electroporation in Intensified
Bioprocessing: Cells Viability and Membrane
Permeabilization
144
5.1.
Introduction
144
5.2.
Material and Methods
150
5.2.1.
Chemicals
150
5.2.2.
Organisms and cultivation
150
5.2.3.
Electroporator and pulse treatment
150
5.2.4.
Viability assay on agar plates
151
5.2.5.
Permeabilization assay by Bleomycin (BLM) treatment
151
5.3.
Results
152
5.3.1.
Effect of voltages, number of pulses and pulses gap on the viability
of M. glutamicus
152
5.3.2.
Effect of growth stages on the viability of M. glutamicus
154
5.3.3.
Effects of different suspending fluids on the viability of
M. glutamicus
157
5.3.4.
Effects of electric pulses on viability of different strains
159
5.3.5.
Assessment of cells electropermeabilization with the aid of Bleomycin
160
5.4.
Discussion
164
6.
Chapter SIX: Osmoregulation of M. glutamicus: Effect of
Hyperosmotic Stress on Its Growth, Viability, L-glutamate
Production and Cytoplasmic Enzymes or Protein Level
173
6.1.
Introduction
173
6.2.
Material and Methods
181
6.2.1.
Chemicals
181
6.2.2.
Organism and cultivation
181
6.2.3.
Microbial growth measurement
181
6.2.4.
Calculation of maximum specific growth rate (µmax)
181
6.2.5.
Hyperosmotic shock of cells suspension of M. glutamicus
182
viii
6.2.6.
Viability assay
182
6.2.7.
Osmoregulation by the addition of compatible solutes
183
6.2.8.
L-glutamate production in M. glutamicus under biotin limited condition
183
6.2.9.
Quantification of glucose, L-glutamate and other amino acids
183
6.2.10. Determination of total protein, GDH and MDH
183
6.3.
Results
184
6.3.1.
Effects of hyperosmotic stress on M. glutamicus growth and viability
184
6.3.2.
Effects of glycine betaine and proline addition on the growth of
hyperosmotically stressed M. glutamicus
6.3.3.
Effects of hyperosmotic stress on L glutamate production by
M. glutamicus
6.3.4.
187
190
Effects of hyperosmotic stress on the activity of enzymes (MDH
and GDH) and total protein
192
6.4.
Discussion
194
7.
Chapter SEVEN: Conclusions and Future Works
200
8.
Chapter EIGHT: References
207
ix
List of Figures
Figures
Title
Page
Figure 2.1
The electron micrograph image of C. glutamicum cell.
Figure 2.2
Structures of a representative mycolic acid from M. tuberculosis
and corynomycolic acid from C. matruchotti
Figure 2.3
10
11
Sugar transport systems of C. glutamicum. PTSGlc, glucose PTS;
PTSFru, fructose PTS; PTSSuc, sucrose PTS; ?, unidentified transport
system. Fru, fructose; Suc, sucrose; Glc, glucose; G6P, glucose
6-phosphate; F6P, fructose 6-phosphate; FBP, fructose-1, 6bisphasphate; Suc6P, sucrose-6-phosphate. The inset shows the
phosphoryltransfer derived from PEP via the general PTS
phosphortransferases I (EI) and HPr proteins shared by the three
substrate specific EII PTS components
Figure 2.4
14
Diagram of the central metabolism of C. glutamicum during growth
on glucose and acetate. Dotted arrows represent pathways consisting
of several reactions, uninterrupted arrows represent single reactions.
AK, acetate kinase; PTA, phosphotransacetylase; ICD, isocitrate
dehydrogenase; ICL, isocitrate lyase; MS, malate synthase; PEPCk,
phosphoenolpyruvate carboxykinase; PCx, pyruvate carboxylase;
PEPCx, phosphoenolpyruvate carboxylase
Figure 2.5
17
Anaplerotic reactions occurring in the central metabolism of C.
glutamicum. Abbreviations used: CoA, coenzyme A; PEP,
phosphoenolpyruvate; PTS phosphotransferase system for glucose
uptake
Figure 2.6
19
Ammonia assimilation or L-glutamate synthesis by the GDH and
GS/GOGAT system. Abbreviations used GDH, glutamate
dehydrogenase; GS, glutamine synthetase; and GOGAT, glutamate
synthase
23
x
Figure 2.7
Schematic drawing of important steps in amino acids production by
bacteria
Figure 2.8
34
A schematic diagram of theoretical cell membrane before and after
Electroporation
Figure 2.9
49
Exposure of a cell to an electric field may result either in
permeabilization of cell membrane or its destruction
Figure 3.1
Simultaneous analysis of glucose and amino acids in fermentation
broth via HPLC
Figure 3.2
75
A correlation between OD and dry weight measurement of M.
glutamicus growth
Figure 3.3a
79
Growth studies of different strains of Corynebacteria on Seed
Medium
Figure 3.3b
79
The growth (OD600) of different strains of Corynebacteria on Seed
Medium
Figure 3.4
80
Effect of different concentrations of biotin on M. glutamicus growth
and glucose consumption
Figure 3.5
82
L-glutamate production in presence of different concentrations of
biotin by M. glutamicus
Figure 3.6
83
L-glutamate production by different strains of Corynebacteria under
biotin limited (1µg l-1) condition
Figure 3.7
88
L-glutamate production by M. glutamicus under different
concentrations of surfactants (added at the start of cultivation)
Figure 3.10
87
Effect of a range of surfactants (added at the exponential phase of
growth) on M. glutamicus growth and glucose consumption
Figure 3.9
84
Effect of a range of surfactants (added at the start of cultivation) on
M. glutamicus growth and glucose consumption
Figure 3.8
51
89
L-glutamate production by M. glutamicus under different
concentrations of surfactants (added at the exponential phase of
growth)
Figure 3.11
89
Effect of a range of concentrations of ethambutol on M. glutamicus
growth and glucose consumption
xi
91
Figure 3.12
L-glutamate production by M. glutamicus due to the exponential
addition of a range of ethambutol concentrations
Figure 3.13
92
L-glutamate production by M. glutamicus fermentation under three
different conditions
93
Figure 4.1
Standard curve of total protein (BSA)
116
Figure 4.2
Standard curve of malate dehydrogenase (MDH)
118
Figure 4.3
Standard curve of glutamate dehydrogenase (GDH)
121
Figure 4.4
Effect of electric field strengths on L-glutamate secretion
(fermentation broth taken after 24h of cultivation)
Figure 4.5
Effect of electric field strengths on L-glutamate secretion
(fermentation broth taken after 48h of cultivation)
Figure 4.6
124
124
Effect of providing pulse gap (30min) and controlling the
temperature of pulsed samples at 4C between two consecutive
pulses on the secretion of L-glutamate (fermentation broth
cultivated for 24h was pulsed at 12.5kV cm-1, 200 and 25µF)
Figure 4.7
126
Effect of providing pulse gap (30min) and controlling the
temperature of pulsed samples at 4C between two consecutive
pulses on the secretion of L-glutamate (fermentation broth cultivated
for 48h was pulsed at 12.5kV cm-1, 200 and 25µF)
Figure 4.8
Effect of electric field strengths on the total protein release by
M. glutamicus
Figure 4.9
126
128
Effect of providing pulse gap (30min) between two pulses (in the
case of multiple pulsing) on the total protein release by
M. glutamicus
Figure 4.10
128
Effect of controlling the temperature of samples at 4C while
allowing 30min pulse gap on the total protein release by M.
glutamicus (pulsed at 12.5kV cm-1, 200 and 25µF)
Figure 4.11
Effect of electric field strengths on the total protein release by
E. coli
Figure 4.12
129
130
Effect of controlling the temperature of samples at 4C while
pulsed on the total protein release by E. coli (field strength, 12.5kV
xii
cm-1 with 30min gap between pulses)
Figure 4.13
Effect of DTT addition followed by pulsation at 12.5kV cm-1,
200 and 25µF on the release of total protein
Figure 4.14
131
132
Release of intracellular proteins of M. glutamicus due to pulse at
12.5kV cm-1, 200 and 25µF (cell pellet, 1g DW was dissolved in
4ml of sterilized distilled water)
Figure 4.15
133
Release of intracellular proteins of M. glutamicus due to sonication,
French press and electroporation (pulsed at 12.5kV cm-1, 200 and
25µF)
134
Figure 5.1
Effect of voltages and multiple pulses on M. glutamicus viability
153
Figure 5.2
Effect of capacitance on the viability of M. glutamicus (cells were
treated by a single pulse)
153
Figure 5.3
Effect of pulse gap or resting time on the viability of M. glutamicus
155
Figure 5.4
Relative cell viability by treating with a range of electric pulses to
the M. glutamicus cells grown at two different growth stages
Figure 5.5
Effect of suspending fluids on the viability of M. glutamicus
(pulsed at 12.5kV cm-1, 200 and 25µF)
Figure 5.6
159
Effect of Bleomycin (5nM) on the viability of M. glutamicus
subjected to a single pulse at 12.5kV cm-1, 200 and 25µF
Figure 5.8
158
Effect of electric pulses on bacterial cell viability (pulsed at 12.5kV
cm-1, 200 and 25µF)
Figure 5.7
156
160
Effect of a range of blemoycin concentrations on the viability
of M. glutamicus exposed to a single pulse at 12.5kV cm-1,
200 and 25µF
Figure 5.9
161
Effect of Bleomycin (5nM) on the viability of M. glutamicus
grown on Seed Medium subjected to multiple pulses at
12.5kV cm-1, 200 and 25µF
Figure 5.10
162
Effect of Bleomycin (5nM) on M. glutamicus viability suspended
in buffer (Tris Buffer 1mM + 270 mM Sucrose) subjected to
multiple pulses at 12.5kV cm-1, 200 and 25µF
Figure 5.11
Effect of Bleomycin (5nM) on the viability of E. coli grown on
xiii
162
Nutrient Medium subjected to multiple pulses at 12.5kV cm-1,
200 and 25µF
163
Figure 6.1
The effects of osmotic stresses on water flux and the turgor pressure
175
Figure 6.2
List of compatibles solutes involved in osmoregulation
175
Figure 6.3
A schematic diagram of the influx and efflux of compatible
solutes during osmoregulation
Figure 6.4
Systems involved during the adaptation of Corynebacteria to
osmotic stresses
Figure 6.5
177
The growth of M. glutamicus in the presence of different
concentrations of NaCl, added at the beginning of cultivation
Figure 6.6
177
184
The growth of M. glutamicus in the presence of different
concentrations of NaCl, added at the exponential phase (7h)
of cultivation
Figure 6.7
185
The reduction in cell viability in the presence of different
strengths of hyperosmotic stress (addition of NaCl at the
start of cultivation)
Figure 6.8
186
The reduction in cell viability in the presence of different
strengths of hyperosmotic stress (addition of NaCl at the
exponential phase, after 7h of inoculation)
Figure 6.9
187
Effect of a range of concentrations of glycine betaine (mM)
on the growth of M. glutamicus grown on hyperosmotic
condition (Seed Medium containing 0.5M NaCl)
Figure 6.10
188
Effect of glycine betaine (20mM, added at start of cultivation)
on the growth of M. glutamicus grown on a range of hyperosmotic
strengths (Seed Medium containing 0.5-1.5M NaCl, added at start
of cultivation)
Figure 6.11
188
Effect of glycine betaine (20mM, exponential phase) on the
growth of M. glutamicus grown on a range of hyperosmotic
conditions (Seed Medium containing 0.5-1.5M NaCl, added
to the exponential growth phase)
Figure 6.12
Effect of a range of concentrations of proline on the growth of
xiv
189
M. glutamicus grown on hyperosmotic condition (Seed Medium
containing 0.5M NaCl)
Figure 6.13
189
Effect of hyperosmotic stress on the growth and glucose
consumption of M. glutamicus grown on biotin limited (1µg l-1)
CGXII Minimal Medium
Figure 6.14
191
Effect of hyperosmotic stress on the production of L-glutamate in
M. glutamicus
Figure 6.15
191
Effect of hyperosmotic stresses on total protein (TP) of M.
glutamicus grown on Seed Medium
Figure 6.16
Effect of hyperosmotic stresses on glutamate dehydrogenase
(GDH) of M. glutamicus grown on Seed Medium
Figure 6.17
192
193
Effect of hyperosmotic stresses on malate dehydrogenase (MDH)
of M. glutamicus grown on Seed Medium
xv
193
List of Tables
Tables
Title
Page
Table 2.1
Features of amino acids production through microbial fermentation
31
Table 2.2
Current production and application of amino acids
32
Table 2.3
Leak Model and Metabolic Flux Change (MFC) Model
42
Table 2.4
Steps of cell electropermeabilization due to electroporation
48
Table 2.5
Critical factors and parameters that influence electroporation
51
Table 2.6
A list of transformation experiments carried out with the different
types of strains by varying the electrical conditions of electroporation
59
Table 3.1
Gradient conditions for analysis of amino acids and carbohydrates
75
Table 3.2
Effect of different concentrations of biotin on specific growth rate
of M. glutamicus
84
xvi