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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. iv 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 vi 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 4C 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 4C 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 4C 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 4C 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