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Pulsed Electric Fields, Page 240 Pulsed electric fields - Influence on physiology, structure and extraction processes of the oleaginous yeast Waltomyces lipofer vorgelegt von Diplom-Biologe Dennis Raschke von der Fakult¨at III - Prozesswissenschaften der Technischen Universit¨at 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 83 Contents 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 effects 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 field 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 Buffers and solutions . 27 3.3.1 Buffers and solutions for analytical methods . 27 3.3.2 Buffers and solutions for lipid extraction . 27 3.4 Cultivationmethods ............................ 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 I 3.6.2 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 field treatment . 49 3.9.1 Generation of pulsed electric fields . 49 3.9.2 Application of pulsed electric field 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 flow 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 Influence of pH and growth rate on the cell size . 74 4.4 Influence of PEF on physiology . 78 4.4.1 Influence of PEF on the structural integrity . 78 4.4.2 Influence of PEF on cell size and vitality . 80 4.4.3 Influence of PEF on the fatty acid pattern . 86 4.5 Influence 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 II 4.6.2 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 Influence of PEF on physiology . 118 5.4 Influence of PEF on extraction processes . 119 6 Conclusions & Perspectives 121 6.1 Growth Monitoring and media optimization . 121 6.2 Influence of PEF on physiology . 121 6.3 Influence of PEF on extraction processes . 122 III List 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 different 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 flow cytometry. 59 4.2 Separation of different vitality subpopulations using flow cytometry . 61 4.3 Cell size and cell shape distribution of W.lipofer ............. 63 4.4 Detection of lipid droplets using flow cytometry . 64 4.5 Oil extraction yields from W.lipofer using different extraction methods and mechanical pretreatments . 66 4.6 Test of different growth media concerning maximum optical density (ODmax), minimal doubling time (td), lipid accumulation as well as cell 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 Influence of pH on cell size of W.lipofer .................. 75 4.11 Correlation between growth rate (µ) and cell size of W.lipofer...... 76 4.12 Proposed scheme for the correlation between dry weight, pH, cell size and lipid droplet development . 77 4.13 Influence of PEF treatment on the structural integrity of W.lipofer .. 78 4.14 Fluorescence microscopy images of untreated and PEF-treated Nile Red stained W.lipofer cells ........................... 79 4.15 Influence of different PEF treatment conditions (2, 3.75 and 25 kV/cm) on the cell size and the vitality of 6 day old W.lipofer cells . 81 IV 4.16 Influence of PEF treatment on the vitality of W.lipofer cells depending oncultureage................................ 84 4.17 Influence of PEF treatment on the fatty acid pattern of W.lipofer ... 86 4.18 Increase of the electric current during PEF treatment of W.lipofer at different conditions . 89 4.19 Development of electric current and vitality during PEF treatment of (A) W.lipofer and (B) S.cerevisiae .................... 90 4.20 Development of the dry weight of W.lipofer due to PEF treatment . 93 4.21 Influence of PEF treatment on the extraction of proteins from W.lipofer 95 4.22 Influence of PEF treatment on the extraction of DNA from W.lipofer . 98 4.23 Comparison of fatty acid composition of Miglyol®812 and 5 day old W.lipofer cells................................ 99 4.24 Fatty acid pattern of untreated W.lipofer cells and cells after PEF treatment at 25 kV/cm and an energy input of 20 kJ/kg with and without extraction with Miglyol®812 for 2 hours. 100 4.25 Extraction of yeast lipids using Miglyol®812 and subsequent continuous PEF treatment. 102 4.26 Extraction of yeast lipids using Miglyol®812 after ultrasound treatment.104 4.27 Drying curves at 60 of untreated and PEF treated W.lipofer cells. 106 4.28 Influence of hot air drying and PEF on the fatty acid distribution of W.lipofer .................................. 107 4.29 Fermentation, PEF treatment and extraction strategies . 109 4.30 Vitality of W.lipofer during growth in suspension culture and integrated batch PEF treatment after 96 h, 120 h and 144 h . 110 4.31 Vitality development in dependence of culture age and PEF treatment during the cultivation of textitW.lipofer . 112 4.32 Comparison of batch and semi-continuous fed-batch fermentation strate- gies for W.lipofer .............................. 115 V List of Tables 3.1 ListofYeasts ................................ 25 3.2 Gentle extraction and analysis procedure . 39 3.3 Fluorescence channels and according dyes . 42 3.4 Flow cytometry detector settings . 43 3.5 Flow cytometry compensation settings . 43 4.1 Refractive indeces of different materials or cell types . 65 4.2 Comparison of batch and continuous PEF treatment conditions . 103 VI Zusammenfassung Im Rahmen der Arbeit wurde der Einfluss der Hochspanungsimpulsbehandlung (HSI) auf die fettbildende Hefe Waltomyces lipofer untersucht. Der Schwerpunkt wurde auf die Extraktion von Proteinen und Fetts¨auren sowie auf die Entwicklung dafur¨ geeigneter Prozessstrategien gelegt. Als Grundlage wurden Methoden zur schnellen Uberwachung¨ von Vitalit¨at und Fetttropfenbildung etabliert, das Medium optimiert, das Wachstumsverhalten dokumentiert und der Einfluss von HSI auf die Struktur und die Physologie von W.lipofer untersucht. Daruber¨ hinaus wurde an Stelle der Anwen- dung von HSI auch eine Ultraschallbehandlung durchgefuhrt.¨ Es wurde getestet, ob mit der Verwendung von Mikroorganismen, energiesparenden Behandlungstech- nologien und dem Verzicht auf organische L¨osungsmittel nachhaltige und umweltfre- undliche Extraktionsprozesse entwickelt werden k¨onnen. Durch die Verwendung von Mikroorganismen kann das gewunschte¨ Produkt in großen Mengen produziert werden ohne auf naturliche¨ Ressourcen, wie z.B. Fisch oder einige Olsaaten,¨ zuruckgreifen¨ zu mussen.¨ Methoden zur schnellen Uberwachung¨ von Vitalit¨at, Zellgr¨oße und der Fettropfen- bidung mittels Durchflusszytomerie und -partikelbildanalyse (Flow particle image analysis - FPIA) wurden erfolgreich etabliert oder angepasst.
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