Project number		Stand-alone Projects  P24154
Title		Identification of Physiological States in Yeast by Novel IR-Techniques
Principal investigator		LENDL Bernhard
Approval date		03.10.2011
University / Research institution		Institut für Chemische Technologien und Analytik, Technische Universität Wien
		Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, Technische Universität Wien
Scientific field(s)
Keywords		in-line bioprocess monitoring, standing MHz ultrasonic waves, FTIR spectroscopy, identification of physiological states of cells
Homepage		http://www.cta.tuwien.ac.at/cavs/

This project aims at developing novel analytical instrumentation and approaches for bioprocess identification and detection of physiological states in yeast cells. An US (ultrasound) enhanced ATR (attenuated total reflection) fiber optic FTIR (Fourier transform infrared) sensor will be further developed and applied for in-line cell analysis. A standing MHz ultrasonic field will allow for positioning of cells in the active region of the ATR diamond and away from it, respectively. Therefore, FTIR spectra of cells only or excreted metabolites (i.e. in the medium) only can be acquired.
S. cerevisiae and P. pastoris will be steered into defined static and transient physiological states, respectively, by different media/environmental limitations. This will lead to differences in storage carbohydrate (SC) (glycogen, trehalose, mannan, etc.) content of the cells, which is strongly related to the type of stress the cells are under. Off-line ATR and transmission FTIR spectra of S. cerevisiae samples in different static physiological states will be acquired and information content will be compared to standard analytical methods, like HPLC and enzymatic essays. Chemometric data analysis will be applied for identification of spectral features related to SC content and latent variables. Thus, the physiological state can be directly determined from FTIR spectra.
Subsequently the US enhanced ATR fiber optic FTIR sensor is coupled in-line to a 20L bioreactor. The same static physiological states as for off-line analysis will be provoked in S. cerevisiae and P. pastoris, limitations will be extended and process condition stress factors like pH and temperature will be introduced. FTIR spectra of the cells and the solutes in the medium only, respectively, will allow for full quantification of stoichiometry and quantification of the primary metabolism. In the final stage of the project, transient physiological states will be provoked by exposing continuous cultures to dynamic changes in extracellular process environment. FTIR spectra acquired in-line with the US enhanced ATR fiber optic FTIR sensor will be analyzed by chemometric techniques for "early-response" spectral markers to physiological changes. As deliverables of the project a tool to detect changes in the physiological state is developed and characterized which can be used for media development and optimization in bioprocess development or process control.