Identification of Physiological States in Yeast by Novel IR-Techniques

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.

Within this project two complementary analytical technologies were combined in a new analyser that now enables the study of microorganism inside a semi-industrial fermenter on a molecular specific level for the first time. Using quasi-standing MHz ultrasound waves it is possible to exert acoustic radiation forces on particles. By doing so, an ultrasonic trap can be configured to capture and pre-concentrate particles. By changing the frequency of the applied MHz sound waves the location where particles actually agglomerate can be shifted in a controlled manner. Such an ultrasonic trap was developed and combined with a commercial mid-IR fibre optic probe for in-line bioprocess monitoring. By measuring mid-IR spectra of the samples, this probe allows to obtain qualitative and quantitative information on the chemical composition of samples which are in close contact to the actual sensor spot, which is actually a planar diamond window of a few square millimetres. Using the described MHz ultrasound technology it is possible to push the particles against the sensor surface or to keep them away from it. Recording mid-IR spectra at these different frequencies and subsequent calculation of the corresponding difference spectra provides access to the mid-IR spectra of the particles (microorganism) only. Upon analysis of these spectra it is possible to obtain qualitative and quantitative information on the chemical composition of the investigated particles. This new analyser was successfully developed, including mechanics, electronics and software and applied for bioprocess monitoring in a stirred, semi-industrial fermenter. As a biochemical model system yeast fermentations were chosen which were performed under different, well defined growth conditions. These conditions were set such that stress due to nutrient limitation was induced. This stress caused a change in the chemical composition of yeast. Especially when setting conditions such that the concentration of sugars (glycogen, trehalose, glucose and mannose) inside the yeast was changing, it was possible to observe these changes in-line. The obtained results were also corroborated by off-line reference analysis using liquid chromatography and again mid-IR spectroscopy. At the end of this project also other fermentations were studied and the performance of different mid-IR in-line probes compared with respect to the achievable signal to noise level. The technology of ultrasound particle manipulation was also combined with other in-line sensor technologies such as Raman spectroscopy and in-line microscopy.