Pulsed electric fields [Elektronische Ressource] : influence on physiology, structure and extraction processes of the oleaginous yeast Waltomyces lipofer / vorgelegt von Dennis Raschke

Informations

• Publié par: technische_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 37
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Pulsed electric elds - In uence on physiology, structure and
extraction processes of the oleaginous yeast
Waltomyces lipofer
vorgelegt von
Diplom-Biologe
Dennis Raschke
von der Fakultat III - Prozesswissenschaften
der Technischen Universitat Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
- Dr.rer.nat -
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dipl.-Ing. Dr. Ulf Stahl
1. Gutachter: Prof. Dr. Dipl.-Ing. Dietrich Knorr
2. Gutachter: Prof. Dr. rer. nat. Sascha Rohn
Tag der wissenschaftlichen Aussprache: 29.09.2010
Berlin 2010
D 83Contents
1 Introduction and objective of the work 1
2 Literature Review 3
2.1 Oleaginous yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.1 Waltomyces lipofer - physiology, morphology and taxonomy . . 4
2.1.2 Lipid metabolism in yeast . . . . . . . . . . . . . . . . . . . . . 5
2.1.3 Fermentation and industrial application of oleaginous yeasts . . 7
2.1.4 Nutritional e ects of polyunsaturated fatty acids . . . . . . . . 9
2.2 Extraction and analysis of yeast lipids . . . . . . . . . . . . . . . . . . 10
2.2.1 Methods for yeast lipid extraction . . . . . . . . . . . . . . . . . 10
2.2.2 Methods for yeast lipid analysis . . . . . . . . . . . . . . . . . . 14
2.3 Single cell proteins (SCP) . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.4 Pulsed electric eld treatment (PEF) . . . . . . . . . . . . . . . . . . . 18
2.4.1 Mechanisms of Electroporation . . . . . . . . . . . . . . . . . . 18
2.4.2 PEF Equipment design . . . . . . . . . . . . . . . . . . . . . . . 19
2.4.3 PEF Process Parameters . . . . . . . . . . . . . . . . . . . . . . 21
2.4.4 Application of PEF . . . . . . . . . . . . . . . . . . . . . . . . . 23
3 Material and Methods 25
3.1 Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.1 YED Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.2 YEG Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.3 Whey permeate (WP) . . . . . . . . . . . . . . . . . . . . . . . 26
3.3 Bu ers and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3.1 Buers and solutions for analytical methods . . . . . . . . . . . 27
3.3.2 Buers and solutions for lipid extraction . . . . . . . . . . . . . 27
3.4 Cultivation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4.1 Maintenance and Storage . . . . . . . . . . . . . . . . . . . . . . 28
3.4.2 Growth in suspension culture . . . . . . . . . . . . . . . . . . . 28
3.4.3 Fermentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.5 Growth measurement using optical density . . . . . . . . . . . . . . . . 30
3.5.1 Stand alone photometer . . . . . . . . . . . . . . . . . . . . . . 30
3.5.2 OD - online monitoring during fermentation . . . . . . . . . . . 31
3.6 Mechanical cell disintegration and extraction . . . . . . . . . . . . . . . 32
3.6.1 Mechanical cell disintegration . . . . . . . . . . . . . . . . . . . 32
I3.6.2 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.6.3 Rotary evaporation . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.7 Lipid extraction and analysis - methodology design and procedure . . . 36
3.7.1 Methodology design . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.7.2 Gentle Extraction and Analysis Procedure . . . . . . . . . . . . 38
3.8 Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.8.1 Water content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.8.2 Flow Particle Image Analysis . . . . . . . . . . . . . . . . . . . 40
3.8.3 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.8.4 Fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . 44
3.8.5 Gas chromatography (GC - FID) . . . . . . . . . . . . . . . . . 45
3.8.6 Protein extraction and analysis . . . . . . . . . . . . . . . . . . 45
3.8.7 Determination of the DNA content . . . . . . . . . . . . . . . . 48
3.8.8 Determination of cell permeabilization by electric current increase 48
3.9 Pulsed electric eld treatment . . . . . . . . . . . . . . . . . . . . . . . 49
3.9.1 Generation of pulsed electric elds . . . . . . . . . . . . . . . . 49
3.9.2 Application of pulsed electric eld treatment . . . . . . . . . . . 51
3.10 Ultrasound treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.10.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.10.2 Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.10.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.11 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4 Results and discussion 58
4.1 Method development for growth monitoring . . . . . . . . . . . . . . . 58
4.1.1 Vitality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.1.2 Lipid droplet development . . . . . . . . . . . . . . . . . . . . . 62
4.1.3 Combination of FPIA and ow cytometry . . . . . . . . . . . . 64
4.1.4 Optimization of lipid extraction methods . . . . . . . . . . . . . 66
4.2 Media optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.3 Growth Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.3.1 Development of OD, cell size, vitality and lipid accumulation . . 71
4.3.2 Inuence of pH and growth rate on the cell size . . . . . . . . . 74
4.4 Inuence of PEF on physiology . . . . . . . . . . . . . . . . . . . . . . 78
4.4.1 Inuence of PEF on the structural integrity . . . . . . . . . . . 78
4.4.2 Inuence of PEF on cell size and vitality . . . . . . . . . . . . . 80
4.4.3 Inuence of PEF on the fatty acid pattern . . . . . . . . . . . . 86
4.5 Inuence of PEF on extraction processes . . . . . . . . . . . . . . . . . 88
4.5.1 Extraction of hydrophilic substances . . . . . . . . . . . . . . . 88
4.5.2 Extraction of lipophilic substances . . . . . . . . . . . . . . . . 99
4.6 Fermentation strategies for PEF and extraction processes . . . . . . . . 109
4.6.1 Batch fermentation with integrated PEF treatment . . . . . . . 110
II4.6.2 Semi continuous fed-batch fermentation with separate PEF treat-
ment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5 Summary 117
5.1 Method development for growth monitoring and media optimization . . 117
5.2 Growth Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
5.3 Inuence of PEF on physiology . . . . . . . . . . . . . . . . . . . . . . 118
5.4 Inuence of PEF on extraction processes . . . . . . . . . . . . . . . . . 119
6 Conclusions & Perspectives 121
6.1 Growth Monitoring and media optimization . . . . . . . . . . . . . . . 121
6.2 Inuence of PEF on physiology . . . . . . . . . . . . . . . . . . . . . . 121
6.3 Inuence of PEF on extraction processes . . . . . . . . . . . . . . . . . 122
IIIList of Figures
2.1 Structure and nomenclature of some omega fatty acids . . . . . . . . . 5
2.2 Proposed model for the formation of lipid droplets . . . . . . . . . . . . 6
2.3 Generalized phase diagram . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4 Mechanism of electropermeabilization . . . . . . . . . . . . . . . . . . . 19
2.5 PEF treatment equipment for the generation of di erent pulse shapes . 20
3.1 Schematic setup of the EloFerm fermentation unit. . . . . . . . . . . . 29
3.2 Fermentation setup for large scale sampling. . . . . . . . . . . . . . . . 30
3.3 Lipid extraction and analysis - methodology design . . . . . . . . . . . 37
3.4 BSA standard curves for the Bradford protein assay . . . . . . . . . . . 47
3.5 Co-linear treatment chamber for continuous PEF applications . . . . . 50
3.6 Setup for the integration of PEF treatment into the fermentation process. 52
3.7 Setup for lab-scale ultrasound treatment of yeast cells . . . . . . . . . . 54
4.1 Determination of vitality using ow cytometry. . . . . . . . . . . . . . . 59
4.2 Separation of di erent vitality subpopulations using ow cytometry . . 61
4.3 Cell size and cell shape distribution of W.lipofer . . . . . . . . . . . . . 63
4.4 Detection of lipid droplets using ow cytometry . . . . . . . . . . . . . 64
4.5 Oil extraction yields from W.lipofer using di erent extraction methods
and mechanical pretreatments . . . . . . . . . . . . . . . . . . . . . . . 66
4.6 Test of di erent growth media concerning maximum optical density
(OD ), minimal doubling time (t ), lipid accumulation as well as cellmax d
shape and cell structure . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.7 Development of optical density and cell diameter during the growth of
W. lipofer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.8 Development of vitality during the growth of W. lipofer. . . . . . . . . 72
4.9 Monitoring of lipid droplet development. . . . . . . . . . . . . . . . . . 73
4.10 Inuence of pH on cell size of W.lipofer . . . . . . . . . . . . . . . . . . 75
4.11 Correlation between growth rate ( ) and cell size of W.lipofer. . . . . . 76
4.12 Proposed scheme for the correlation between dry weight, pH, cell size
and lipid droplet development . . . . . . . . . . . . . . . . . . . . . . .