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Development of a continuous parallel shaken bioreactor system in small scale [Elektronische Ressource] / vorgelegt von Ali Akgün

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190 pages
Biochemical Engineering ● RWTH Aachen University Ali Akgün DEVELOPMENT OF A CONTINUOUS PARALLEL SHAKEN BIOREACTOR SYSTEM IN SMALL SCALE Development of a Continuous Parallel Shaken Bioreactor System in Small Scale Von der Fakultät für Maschinenwesen der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Ali Akgün aus Usak /Türkei Berichter: Universitätsprofessor Dr.-Ing. Jochen Büchs Universitätsprofessor Dr. rer. nat. habil. Ulrich Klinner Tag der mündlichen Prüfung: 13.07.2007 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. DEDICATION This thesis is dedicated to my future family. My belief in the significance of this dissertation for their life has made this project possible. Preface This dissertation presents the results of my PhD study carried out from October 2001 to February 2005 at the Department of Biochemical Engineering of RWTH Aachen University in Germany. My PhD was financially supported through a scholarship of German Environment Foundation (Deutscher Bundesstiftung Umwelt). I would like to thank my supervisor, Prof. Dr.-Ing. Jochen Büchs (Dept.
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Biochemical Engineering ● RWTH Aachen University







Ali Akgün


DEVELOPMENT OF A CONTINUOUS
PARALLEL SHAKEN BIOREACTOR SYSTEM
IN SMALL SCALE















Development of a Continuous
Parallel Shaken Bioreactor System
in Small Scale




Von der Fakultät für Maschinenwesen der Rheinisch-Westfälischen
Technischen Hochschule Aachen zur Erlangung des akademischen
Grades eines Doktors der Naturwissenschaften genehmigte
Dissertation

vorgelegt von

Ali Akgün
aus
Usak /Türkei



Berichter:
Universitätsprofessor Dr.-Ing. Jochen Büchs
Universitätsprofessor Dr. rer. nat. habil. Ulrich Klinner

Tag der mündlichen Prüfung: 13.07.2007


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.










DEDICATION

This thesis is dedicated to my future family.
My belief in the significance of this dissertation for their life
has made this project possible.

















Preface

This dissertation presents the results of my PhD study carried out from October 2001 to
February 2005 at the Department of Biochemical Engineering of RWTH Aachen University
in Germany. My PhD was financially supported through a scholarship of German
Environment Foundation (Deutscher Bundesstiftung Umwelt).

I would like to thank my supervisor, Prof. Dr.-Ing. Jochen Büchs (Dept. of Biochemical
Engineering, RWTH Aachen University), for the opportunity to carry out this PhD study and
especially for introducing me kindly to the engineering aspects of biotechnology, and Prof.
Dr. rer. nat. Ulrich Klinner (Institute for Biology IV, RWTH Aachen University) and Prof.
Dr.-Ing. Andreas Pfenning (Dept. of Chemical Engineering, RWTH Aachen University) for
their contributions as a member of my examination committee.

I wish to thank the German Environment Foundation for financial support during my PhD.

I am also grateful to Dr.-Ing. Okan Akin for sharing his enthusiastic attitude towards
academic career, and for his special interest in my personal and professional development in
Germany

Special thanks go to Dr. Nursen Sözer for her kindly help and guidance during my study in
Germany, to all my colleagues, personnel and friends at the Department of Biochemical
Engineering of RWTH Aachen University for a warm and friendly atmosphere, and to all my
friends for their valuable friendship and encouragement for making life in aboard bearable.

Finally, my deepest gratitude is extended to my parents and my uncle for their love and
support sent across a half globe to encourage me to continue my endeavor, and to make this
step in my life possible.



July, 2007
Ali Akgün


V



Contents



Kurzfassung IX

Abstract XI

1 General Introduction 1
1.1 Background….………………………………………………………………. 1
1.2 Continuous-Flow Cultivation….…………………………………………….. 3
1.2.1 Overview of the Continuous-Flow Cultivation...…...……………...... 3
1.2.2 Conventional Bioreactors for Continuos-Flow.……….…………...... 7
1.3 Motivation and aim…………………………………………………….......... 11
2 Theoretical Background of Shaking Technology 15
2.1 Shaking Machine…………………………………………………………….. 15
2.2 Shake Vessels………………………………………………………………... 21
2.3 Characteristics of Fluid in Unbaffled Shaken Flasks…...………………….... 26
2.3.1 Hydrodynamic in unbuffled Shaken Flasks………………………..... 26
2.3.1.1 Types of Fluid Behavior……..…..……………………........ 26
2.3.1.2 Dimensionless Numbers………………………………........292.3.1.3 Liquid Distribution in Shaken Flask…………………......... 33
2.3.2 Gas Exchange and Aeration in Shaken Flasks………………………. 34
2.3.2.1 Gas Diffusion through sterile plug……………………........ 35
2.3.2.2 Gas-Liquid Mass Transfer……………………………......... 352.3.2.3 Consideration of Oxygen Transfer Rate (OTR)…………… 40
2.4 Overview of Microbial Growth in Shaking Culture…………………………. 44

3 Fundamentals of Continuous-Flow Culture 53
4 Materials and Methods 59
VI

4.1 250 – mL Erlenmeyer Flask….……………………….…...……………........ 59
4.2 Materials Considered for Construction………………………...……………. 59
4.3 0.5 M Sulphite System………………………………………………………. 61
4.4 On-line Measurement of Oxygen Transfer Capacity………………………... 63
4.5 Biological Systems ………….………………...…………………………….. 63
4.5.1 Saccharomyces cerevisiae (ATCC 32167)….……………………...... 63
4.5.2 Corynebacterium glutamicum (ATC 13032)………………………… 65
4.5.3 Pichia stipitis (CBS 5774)………………….………………………... 66
4.6 Analytic Methods……………………………………………………………. 66
5 Principle of Continuous
Parallel Shaken Bioreactor System and Prototyp-I 69
5.1 Continuous Parallel Shaken Bioreactor System………...…………………… 70
5.1.1 Principle and Set-up of CosBios-I….………………………………... 70
5.1.2 Construction Elements of CosBios-I...………………………………. 71
5.2 Overview of Experimental Set-up..….………………………………………. 72
5.2.1 Technical Characterisation……...….………………………………... 725.2.2 Biological Validation……………......………………………………. 74
5.3 Results and Discussions……………………………………………………... 76
5.3.1 Results of Technical Characterisation...……………………………... 76
5.3.1.1 Influence of Aeration Rate on Filling Volume…………….. 76
5.3.1.2 Influence of Substrate Feeding Rate on Filling Volume..…. 76
5.3.1.3 Influence of Shaking Diameter on Filling Volume ……….. 77
5.3.1.4 Influence of Outlet Height on Filling Volume…………….. 78
5.3.1.5 Influence of Shaking Frequency on Filling Volume………. 79
5.3.1.6 Comparison with Model Calculations...…………………… 79
5.3.2 Results of Biological Validation...…………………………………... 80

6 Improved Continuous
Parallel Shaken Bioreactor System Prototyp-II 83
6.1 Improved Bioreactor Vessel of CosBios...…..………………………………. 83
6.2 Set-up of CosBios-II.......…………………………………………………….. 85
6.3 Significance of Oxygen Supply……………………………………………… 86
6.4 Experimental Set-up…………………………………………………………. 87
6.4.1 Technical Characterization…………………………………………... 87