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J Schnöller, J Baena, G Mazarevica, B. Adamitsch, A. Kainz, B. Lendl:
"DEVELOPMENT AND APPLICATION OF A PORTABLE FTIR-ATR ANALYSER FOR ON-LINE MONITORING OF MICRO-ORGANISM DURING BIOPROCESSES ";
Poster: ICAVS II, Nottingham (UK); 19.08.2003 - 24.08.2003.

In this contribution a strategy for reliable on-line monitoring of micro-organism during bio-processes is presented on the examples of yeast and E. coli fermentations. An automated flow system is connected via a fast loop to the fermentor. From the fast loop small volumes of fermentation broth are taken by the automate flow system and pumped through a home made flow cell which incorporates an horizontal attenuated total reflection (ATR) unit (Durasample II, SensIR). The ATR cell was attached to a portable Bruker IR-cube. As long as the flow is maintained spectra dominated by dissolved reagents and products (glucose, ethanol, organic acids,...) can be recorded. However, upon stopping the flow the cells present in the fermentation broth settle on top of the ATR surface thus coming into reach of the evanescent field, which allows to record FTIR spectra of the cells. Using this technique changes in the composition of the cells during the bio-process can be followed on-line. An important feature of the developed experimental set-up is the possibility to flush the ATR surface with rinsing solutions (NaHCO3, dist. H2O) using the flow systems without interfering the bio-process to be monitored. In this way bio-films, which are unavoidably formed on the ATR surface upon prolonged contact with the fermentation broth, can be removed and thus their interference on the measurement avoided. Furthermore flushing with distilled water also allows recording of a new background spectrum if required.We used this technique to monitor yeast cells during a fed-batch fermentation that started with an exponential growth phase (4 generation cycles) and continued with a linear maturation phase under nitrogen limitation. From the recorded spectra the change in the protein-carbohydrate ratio could be followed directly. Furthermore, using second derivative spectra different carbohydrates (glycogen, trehalose, mannan) could be distinguished and their different rates of formation in the cells during the maturation phase determined.In case of poly-hydroxybutyric acid (PHB) production by an E coli fermentation the developed set-up could be used to follow product formation on-line. PHB, which shows a characteristic C=O band, is enriched in the E.coli cells during the bio-process. Results of quantitative off-line, reference analysis of the produced PHB showed excellent correlation with the intensity of the recorded C=O band of the analyte present in the E. coli cells.