In Situ Fluorescence Cell Mass Measurements of Saccharomyces cerevisiae Using Cellular Tryptophan

Abstract

This work describes a new spectroscopic optical fiber/rod technique for in situ real time measurement of cell mass and product concentrations in bioreactors using intrinsic fluorescence. The variable excitation/emission wavelength capability of this sensor allows for species-selective measurement during fermentations. Cell mass (tryptophan) and product concentrations (pyridoxine) have been measured during fermentations of Saccharomyces cerevisiae. The effects of varying substrate concentration and oxygen concentration on the observed cell mass signals are eliminated by direct measurement of cell mass, as opposed to indirect measurement schemes such as those using NADH fluorescence. The sensor is robust and able to undergo many cycles of in situ steam sterilization without degradation, and its fluorescence signal is linear with concentration for all species studied in this work. Tryptophan fluorescence from yeast is shown to be a better measure of cell mass than NADH fluorescence.

In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures

“…Based on literature it was possible to associate some regions of this map to specific fluorophors, namely Phe, Tyr, Trp , pyridoxine, and riboflavin Marose et al, 1998;García et al, 2001). In bioreaction culture bulks, the maximum fluorescence emission for Trp normally reported corresponds to excitation at 290 nm Horvath et al, 1993;Surribas et al, 2006c). However, the maximum fluorescence signal for each fluorophor changes with the environmental conditions .…”

“…Fluorescence signal from intracellular proteins (Horvath et al, 1993) and from vitamins (Hisiger and Jolicoeur, 2005a) have been successfully used to monitor microbial growth. The fluorescence of NAD(P)H has been used preferentially for monitoring cellular activity since their concentration within cells depends on cells' metabolic state Tartakovsky et al, 1996;Marose et al, 1998).…”

“…Mammalian cells are not able to synthesize vitamins and none of the fluorescent amino acids present in the medium. Therefore, cellular growth can not be directly correlated with amino acids or vitamins fluorescence, as in the majority of microbial fermentations (Horvath et al, 1993;Hisiger and Jolicoeur, 2005a). In such cases, multivariate chemometric techniques can be used to analyze the whole spectra.…”

Bioanalytics: detailed insight into bioprocesses

“…On the basis of these experiences, miniaturized fluorimetric probes were built [28][29][30]41] mainly for the monitoring of the NAD(P)H dependent fluorescence. Only few systems were described where different wavelenghts could be monitored [30,42,43]. Even when these techniques offer the possibility to non-invasively monitor the metabolic state of the cultivated microorganisms, the signal interpretation was often difficult.…”

Biomass estimation using fluorescence measurements in Pichia pastoris bioprocess

“…13 -16 It has been reported that the tryptophan fluorescence signal from intracellular protein structures in Saccharomyces cerevisiae can be used to monitor cell growth. 17 This shows some advantages over NADH fluorescence, as it is not so dependent upon the cell's metabolic state. Other works have analysed the possibilities of fluorometry for monitoring purposes in bioprocesses yielding a moderate cell density level.…”