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6 7 Integrated Sensor Array for On-Line Monitoring 6 7 INTEGRATED SENSOR ARRAY FOR ON‐LINE MONITORING MICRO BIOREACTORS The described research has been carried out at the BIOS Lab‐on‐a‐Chip group of the MESA+ Institute for Nanotechnology of the University of Twente, Enschede, The Netherlands. The research was financially supported by the Dutch Science Foundation (NWO) through the ACTS program IBOS (Integration of Biosynthesis and organic synthesis), DSM Anti‐Infectives, Organon and Applikon. Samenstelling promotiecommissie: voorzitter: prof. dr. W.H.M. Zijm secretaris: prof. dr. ir. A.J. Mouthaan promotor: prof. dr. ir. A. van den Berg assistent promotor: prof. dr. J.G.E. Gardeniers leden: prof. dr. P. Renaud prof. dr. ir. P.H. Veltink prof. dr. I. Vermes dr. H.J. Noorman prof. dr. ir. J.J. Heijnen Title: Integrated sensor array for on‐line monitoring micro bio reactors Author: Erik Krommenhoek ISBN: 978‐90‐365‐2593‐0 Printing: PrintPartners Ipskamp Copyright © 2007 by Erik Krommenhoek, Enschede, The Netherlands 8 9 INTEGRATED SENSOR ARRAY FOR ON‐LINE MONITORING MICRO BIOREACTORS PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit Twente, op gezag van rector magnificus, Prof. Dr. W.H.M. Zijm, volgens het besluit van het College voor Promoties in het openbaar te verdedigen op donderdag 29 november 2007 om 13.15 uur door Erik Eduard Krommenhoek geboren op 29 juli 1979 te Apeldoorn Dit proefschrift is goedgekeurd door: promotor: prof. dr. ir. Albert van den Berg co‐promotor: prof. dr. Han Gardeniers Contents Contents Chapter 1: Scope and outline of this thesis .................................................................. 5 1.1 Introduction...............................................................................................................5 1.2 Yeasts in biotechnology.............................................................................................6 1.3 Research goal ............................................................................................................9 1.4 Outline of this thesis................................................................................................10 References.....................................................................................................................11 Chapter 2: Exploration of miniaturized fermentation monitoring techniques .............13 2.1 Introduction.............................................................................................................13 2.2 Biosensor fundamentals..........................................................................................14 2.2.1 Optical sensors .................................................................................................15 2.2.2 Electrochemical sensors...................................................................................17 2.3 Fermentation monitoring........................................................................................21 2.3.1 pH .....................................................................................................................22 2.3.2 Dissolved oxygen..............................................................................................25 2.3.3 Biomass.............................................................................................................28 2.3.4 Temperature.....................................................................................................28 2.4 Experimental ...........................................................................................................29 2.4.1 pH .....................................................................................................................29 2.4.2 Dissolved oxygen..............................................................................................32 2.4.3 Biomass.............................................................................................................34 2.4.4 Temperature.....................................................................................................34 2.5 Measurement results ..............................................................................................34 2.5.1 pH .....................................................................................................................35 2.5.2 Dissolved oxygen..............................................................................................38 2.5.3 Biomass.............................................................................................................41 2.6 Discussion & conclusions.........................................................................................42 References.....................................................................................................................44 Chapter 3: Monitoring of yeast cell concentration using a micromachined impedance sensor .........................................................................................................................51 3.1 Introduction.............................................................................................................51 3.2 Theory......................................................................................................................52 3.3 Experimental ...........................................................................................................55 3.3.1 Device fabrication.............................................................................................55 3.3.2 Measurement procedures................................................................................56 3.4 Measurement results ..............................................................................................57 3.5 Conclusion ...............................................................................................................61 References.....................................................................................................................62 1 Contents Chapter 4: Integrated electrochemical sensor array for on‐line monitoring of yeast fermentations.............................................................................................................65 4.1 Introduction.............................................................................................................65 4.2 Theory......................................................................................................................67 4.2.1 Biomass.............................................................................................................67 4.2.2 Dissolved oxygen..............................................................................................70 4.2.3 pH .....................................................................................................................72 4.2.4 Temperature.....................................................................................................73 4.3 Experimental ...........................................................................................................74 4.3.1 Sensor design ...................................................................................................74 4.3.2 Device fabrication.............................................................................................75 4.3.3 Measurement setup.........................................................................................76 4.4 Results .....................................................................................................................77 4.4.1 Biomass measurement results .........................................................................77 4.4.2 Dissolved oxygen measurement results...........................................................78 4.4.3 pH measurement results..................................................................................79 4.4.4 Temperature measurement results .................................................................80 4.5 Conclusions..............................................................................................................80 References..................................................................................................................... 81 Chapter 5: Lab‐scale fermentation tests of micro chip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration.....85 5.1 Introduction.............................................................................................................85 5.1.1 pH .....................................................................................................................87 5.1.2 Dissolved oxygen concentration ......................................................................88 5.1.3 Biomass concentration.....................................................................................88 5.1.4 Temperature.....................................................................................................89 5.1.5 Sensor integration ............................................................................................89 5.2 Materials and methods ...........................................................................................90 5.2.1 Design and fabrication of the multi‐sensor chip..............................................90 5.2.2 Readout of the sensor chip ..............................................................................91 5.2.3 Batch and continuous cultivation.....................................................................91
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