In vivo kinetics with rapid perturbation experiments in Saccharomyces cerevisiae using a second-generation BioScope.

We present a robust second-generation BioScope: a system for continuous perturbation experiments. Firstly, the BioScope design parameters (i.e., pressure drop, overall oxygen (O2) and carbon dioxide (CO2) mass transfer, mean residence time distribution and plug flow characteristics) were evaluated. The average overall mass transfer coefficients were estimated to be 1.8E-5 m s(-1) for O2 and 0.34E-5 m s(-1) for CO2. It was determined that the O2/CO2 permeable membrane accounted for 75% and 95% of the overall resistance for O2 and CO2, respectively. The Peclet number (Pe) of the system was found to be >500 for liquid flow rates between 1 and 4 ml min(-1), ensuring plug flow characteristics. Secondly, steady-state intracellular metabolite concentrations obtained using direct rapid sampling from the fermentor were compared with those obtained by rapid sampling via the pre-perturbation sample port of the BioScope. With both methods the same metabolite levels were obtained. Thirdly, glucose perturbation experiments were carried out directly in the fermentor as well as in the BioScope, whereby steady-state Saccharomyces cerevisiae cells from a glucose/ethanol limited chemostat were perturbed by increasing the extracellular glucose concentration from 0.11 to 2.8 mM. Intracellular and extracellular metabolite levels were measured within a time window of 180 s. It was observed that the dynamic metabolite concentration profiles obtained from both perturbations were nearly the same, with the exception of the C4 metabolites of the TCA cycle, which might be due to differences in culture age.