Process Concept and Designs for Value Added Chain of the Phototrophic Algae Chlamydomonas

156 Pages·2017·3.84 MB·English

by  Joachim Hahm

Process Concept and Designs for Value
Added Chain of the Phototrophic Algae
Chlamydomonas asymmetrica

Verfahrenskonzept und Entwürfe für die Wertschöpfungskette
der phototrophen Alge Chlamydomonas asymmetrica

Der Technischen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg

Zur Erlangung des Doktorgrades
Dr.-Ing.

vorgelegt von
Joachim Hahm

aus Erlangen
Als Dissertation genehmigt
von der Technischen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 16. Juli 2018

Vorsitzender des Promotionsorgans: Prof. Dr.-Ing. Reinhard Lerch
Gutachter: Prof. Dr. rer. nat. Rainer Buchholz
Prof. Dr.-Ing. Cornelia Rauh
Acknowledgement
I want to thank Prof. Rainer Buchholz for giving me the opportunity and the trust and the
support to work for him at the chair of bioprocess engineering in Germany and in South Korea.
The many discussions and advice were a great support for completing my dissertation.
Furthermore, I want to thank Prof. Cornelia Rauh as the second reviewer, Prof. Monika
Pischetsrieder as the authorised examiner and Prof. Antonio Delgado as the chairperson for
their support in the exam committee.
I want to thank my colleagues at the department in Germany, especially, Katja Steinbach and
Ingrid Callies for their never-ending intercontinental support regarding all kinds of matters.
Furthermore, I want to thank my scientific companions Sascha Baer and Josephine Taucher for
all the teamwork including helpful advices and discussions.
Sincere thanks go to the members of the Flying Faculty of the FAU Busan Branch Campus
whose number is too high to be listed here. I appreciated the many insights in other fields of
science and the personal contact including general talks and discussions with many of you.
Astrid Lindner, Eunhye Im, Andreas Bauer, Sanghun Yun, and all the other students thank you
very much for the great time we had working together in the B-lab. I will never forget!
Without the staff of the FAU Busan Campus Sung-Ho Beak, Heejae, Yang, Huichih Wang and
all others, this work would have been hardly possible. I have to emphasize my sincere gratitude
to Katharina Sommer for her immense helpfulness and support over the last years. Furthermore,
I want to thank Dr. José Rodríguez Agudo, Dr. Giovanni Luzi, Dr. Soebiakto Loekman, Dr.
Matthias Reichert, Sun Hwa Jung, Marc Williams and all other former co-workers who helped
to keep the laboratories running and who were always there for me. Thank you very much!
Dr. Christoph Lindenberger made me come back to Korea twice. Without his trust, guidance,
knowledge and especially friendship I would not be where I am right now. Thank you for eve-
rything. You will always be a welcome guest in “Café Hahm”!
Finally, I have to express my infinite gratefulness to my family and particularly my wife who
supported me immensely over the last few years. Your support kept me running all the time. It
is difficult to find words to express my feelings but I can ensure you with true heart that I will
never forget what you have done for me!

Table of Content - I -

Table of Content

Table of Content ...................................................................................................................................... I
List of Abbreviations ............................................................................................................................. V
List of Symbols ................................................................................................................................... VII
Zusammenfassung ................................................................................................................................. IX
Abstract .................................................................................................................................................. X
1 Introduction ..................................................................................................................................... 1
2 State of the Art ................................................................................................................................ 3
2.1 Diversity of Phototrophic Microorganisms ............................................................................. 3
2.2 Microalgae Metabolism .......................................................................................................... 5
2.2.1 Photosynthesis ................................................................................................................. 5
2.2.2 Dark Respiration ............................................................................................................. 7
2.3 Microalgae Derived Products .................................................................................................. 8
2.3.1 Fatty Acids ...................................................................................................................... 9
2.3.2 Carbohydrates ............................................................................................................... 11
2.3.3 Chlorophylls .................................................................................................................. 13
2.3.4 Carotenoids ................................................................................................................... 14
2.3.5 Others ............................................................................................................................ 16
2.4 Considerations for the Cultivation of Microalgae ................................................................. 16
2.4.1 Microorganism Selection .............................................................................................. 17
2.4.2 Light Requirements ....................................................................................................... 17
2.4.3 Reactor Systems ............................................................................................................ 21
2.4.4 Reactor Process Management ....................................................................................... 23
2.4.5 Downstream Processing ................................................................................................ 25
2.4.6 Biorefinery Concepts .................................................................................................... 25
2.5 Successfully Implemented Algae Processes ......................................................................... 26
2.5.1 Biomass Production ...................................................................................................... 26
2.5.2 β-Carotene Production .................................................................................................. 26
2.5.3 Astaxanthin Production ................................................................................................. 27
- II - Table of Content

3 Problem Statement ........................................................................................................................ 28
4 Material and Methods ................................................................................................................... 29
4.1 Materials ............................................................................................................................... 29
4.1.1 Chemicals ...................................................................................................................... 29
4.1.2 Equipment ..................................................................................................................... 30
4.1.3 Organisms ..................................................................................................................... 31
4.2 Methods................................................................................................................................. 32
4.2.1 Culture Maintenance ..................................................................................................... 32
4.2.2 Medium Preparation ...................................................................................................... 32
4.2.3 Splitting ......................................................................................................................... 33
4.2.4 Inoculum Preparation .................................................................................................... 33
4.2.5 Upstream Process Monitoring ....................................................................................... 33
4.2.5.1 Dry Weight/ Concentration ....................................................................................... 33
4.2.5.2 Optical Density ......................................................................................................... 33
4.2.5.3 Inlet and Outlet Gas Stream/Composition ................................................................ 34
4.2.5.4 Flow and Dilution Rate ............................................................................................. 34
4.2.5.5 Light Intensity ........................................................................................................... 34
4.2.5.6 Culture pH, DO and Temperature ............................................................................. 35
4.2.5.7 Medium Nitrate Content ........................................................................................... 35
4.2.6 Downstream Process Monitoring .................................................................................. 35
4.2.6.1 Biomass Harvest ....................................................................................................... 35
4.2.6.2 Biomass Lyophilization ............................................................................................ 36
4.2.6.3 Pigments Analysis ..................................................................................................... 36
4.2.6.4 Fatty Acid Analysis ................................................................................................... 37
4.2.6.5 Total Carbohydrate Analysis .................................................................................... 38
4.3 Reactor Systems .................................................................................................................... 38
4.3.1 Reactor Types ............................................................................................................... 38
4.3.2 Reactor Illumination ..................................................................................................... 40
4.3.3 Reactor System Setups .................................................................................................. 41
Table of Content - III -

5 Results ........................................................................................................................................... 44
5.1 Basic Characterization of C. asymmetrica ............................................................................ 44
5.1.1 Medium-Screening ........................................................................................................ 44
5.1.2 Temperature-Optimum .................................................................................................. 46
5.1.3 Light-Optimum ............................................................................................................. 48
5.1.4 Reactor Type ................................................................................................................. 52
5.2 Detailed Characterization of C. asymmetrica: Chemostat .................................................... 56
5.2.1 Growth Related Parameters .......................................................................................... 57
5.2.2 High Value Products ..................................................................................................... 58
5.2.3 Nitrate Concentration Variation .................................................................................... 64
5.3 Cascade System .................................................................................................................... 67
5.3.1 General Idea .................................................................................................................. 67
5.3.2 Cascade Studies............................................................................................................. 67
5.3.3 Cascade Optimization ................................................................................................... 79
5.3.4 Cascade: Medium Recycling ........................................................................................ 82
6 Significance Analysis .................................................................................................................... 85
6.1 Statistical Analysis ................................................................................................................ 85
6.2 Culture Preparation ............................................................................................................... 86
6.3 Cultivation............................................................................................................................. 87
6.4 Culture Analysis .................................................................................................................... 89
7 Discussion ..................................................................................................................................... 92
7.1 Selection of the Production Organism .................................................................................. 92
7.2 Basic Parameters for Batch Cultivation of C. asymmetrica .................................................. 92
7.2.1 Medium ......................................................................................................................... 93
7.2.2 Cultivation Temperature ............................................................................................... 94
7.2.3 Light requirements ........................................................................................................ 95
7.3 Reactor Geometry Variation ................................................................................................. 96
7.4 Chemostat Cultivation of C. asymmetrica .......................................................................... 101
7.4.1 Impact of Continuous Process on High Value Product Accumulation ....................... 101
- IV - Table of Content

7.4.2 Comparison of Continuous and Batch Systems. ......................................................... 106
7.5 Two-Stage Reactor System ................................................................................................. 107
7.6 Process Design Impact on Down Stream ............................................................................ 113
7.7 Biorefinery Process ............................................................................................................. 114
8 Outlook ....................................................................................................................................... 115
9 Bibliography ............................................................................................................................... 117
10 Appendix ................................................................................................................................. 127
10.1 Supplementary Figures and Tables ..................................................................................... 127
10.2 Analytical Methods and Calibrations .................................................................................. 132
10.2.1 HPLC Method and Calibration ................................................................................... 132
10.2.2 GC-MS Method and Calibration ................................................................................. 133
10.2.3 Nitrate Calibration....................................................................................................... 134
10.2.4 Carbohydrate (EPS) Calibration ................................................................................. 134
10.3 Media List ........................................................................................................................... 135
10.4 List of Figures ..................................................................................................................... 140
10.5 List of Tables ...................................................................................................................... 142
List of Abbreviations - V -

List of Abbreviations
Abbreviation Meaning
ADP Adenosine Diphosphate
ALA 5-Aminolevulinic Acid
AS Asthanxathin
ATP Adenosine Triphosphate
BC β-Carotene
CA Compressed Air
CBB Calvin-Benson-Bassham cycle
CCM Carbon Concentrating Mechanism
CHA Chlorophyll a
CHB Chlorophyll b
COD Coefficient of Determination
DCM Dichloromethane
dH O Deionised Water
2
DHA Docosahexaenoic Acid
DIW Deionised Water
EPA Eicosapentaenoic Acid
EPS Extracellular Polysaccharides
ER Endoplasmatic Reticulum
FA Fatty Acid
FADH Flavin Adenine Dinucleotide
FAME Fatty Acid Methyl Ester
FPA Flat Panel Airlift
G-3-P Glyceraldehyde-3-Phosphate
GC-MS Gas Chromatography–Mass Spectrometry
GGPP Geranyl Pyrophosphate
GLA γ-Linoletic Acid
GLP Good Laboratory Practice
GMP Good Manufacturer Practice
HBR Hanging Bag Reactor
HCMV Human Cytomegalie Virus
HPLC High Performance Liquid Chromatography
- VI - List of Abbreviations

HVP High Value Products
I Compensation Point
C
I Inhibition Point
I
I Saturation Point
K
IPP Isopentenyl Pyrophosphate
IS Internal Standard
KHV Koi Herpesvirus
LCA Life Cycle Assessment
LED Light Emitting Diode
LU Lutein
MTBE Tert-Butyl Methyl Ether
NADH Nicotinamide Adenine Dinucleotide
NADPH Nicotinamide Adenine Dinucleotide Phosphate
OD Optical Density
PAR Photosynthetically Active Radiation
PC Polycarbonate
PFD Photon Flux Density
PS I Photosystem I
PS II Photosystem II
PSM Photobioreactor Screening Module
PUFA Polyunsaturated Fatty Acid
PVC Polyvinyl Chloride
R1 Reactor/Stage one
R2 Reactor/Stage two
RGB Red, Green and Blue
RT Room Temperature
RZA Raum-Zeit-Ausbeute
TAG Triglyceride
TEP Transparent Exoploymer Particles
UV/VIS Ultraviolet/Visible Part of Light Spectrum
VHSV Viral Hemorrhagic Septicaemia Virus