In this research, two dynamic (13)C-labeling experiments confirmed turnover and rapid mobilization of stored glycogen and trehalose in an aerobic glucose-limited chemostat (D=0.05 h(-1)) culture of Saccharomyces cerevisiae. In one experiment, the continuous feed to an aerobic glucose-limited chemostat culture of S. cerevisiae was instantaneously switched from naturally labeled to fully (13)C labeled while maintaining the same feed rate before and after the switch. The dynamic replacements of naturally labeled intracellular glycolytic intermediates and CO(2) (in the off-gas) with their (13)C-labeled equivalents were measured. The data of this experiment suggest that the continuous turnover of glycogen and trehalose is substantial (c. 1/3 of the glycolytic flux). The second experiment combined the medium switch with a shiftup in the glucose feeding rate (dilution rate shiftup from 0.05 to 0.10 h(-1)). This experiment triggered a strong but transient mobilization of storage carbon, that was channelled into glycolysis, causing a significant disruption in the dynamic labeling profile of glycolytic intermediates. The off-gas measurements in the shiftup experiment confirmed a considerable transient influx of (12)C-carbon into glycolysis after the combined medium switch and dilution rate shiftup. This study shows that for accurate in vivo kinetic interpretation of rapid pulse experiments, glycogen and trehalose metabolism must be taken into account.

In this study, a previously developed mini-bioreactor, the Biocurve, was used to identify an informative stimulus-response experiment. The identified stimulus-response experiment was a modest 50% shift-up in glucose uptake rate (qGLC) that unexpectedly resulted in a disproportionate transient metabolic response. The 50% shift-up in qGLC in the Biocurve resulted in a near tripling of the online measured oxygen uptake (qO 2 ) and carbon dioxide production (qCO 2 ) rates, suggesting a considerable mobilization of glycogen and trehalose. The 50% shift-up in qGLC was subsequently studied in detail in a conventional bioreactor (4 l working volume), which confirmed the results obtained with the Biocurve. Especially relevant is the observation that the 50% increase in glucose uptake rate led to a three-fold increase in glycolytic flux, due to mobilization of storage materials. This explains the unexpected ethanol and acetate secretion after the shift-up, in spite of the fact that after the shift-up the qGLC was far less than the critical value. Moreover, these results show that the correct in vivo fluxes in glucose pulse experiments cannot be obtained from the uptake and secretion rates only. Instead, the storage fluxes must also be accurately quantified. Finally, we speculate on the possible role that the transient increase in dissolved CO 2 immediately after the 50% shift-up in qGLC could have played a part in triggering glycogen and trehalose mobilization.

A mini bioreactor (3.0 mL volume) has been developed and shown to be a versatile tool for rapidly screening and quantifying the response of organisms on environmental perturbations. The mini bioreactor is essentially a plug flow device transformed into a wellmixed reactor by a recycle flow of the broth. The gas and liquid phases are separated by a silicone membrane. Dynamic mass transfer experiments were performed to determine the mass transfer capacities for oxygen and carbon dioxide. The mass transfer coefficients for oxygen and carbon dioxide were found to be 1.55 AE 0.17 Â 10 À5 m/ s and 4.52 AE 0.60 Â 10 À6 m/s, respectively. Cultivation experiments with the 3.0 mL bioreactor show that (i) it can maintain biomass in the same physiological state as the 4.0 L lab scale bioreactor, (ii) reproducible perturbation experiments such as changing substrate uptake rate can be readily performed and the physiological response monitored quantitatively in terms of the O 2 and CO 2 uptake and production rates. ß