New bioreactor‐coupled rapid stopped‐flow sampling technique for measurements of metabolite dynamics on a subsecond time scale

Abstract

Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks. As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation. The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor. For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed. A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber. Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified. The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli.

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

“…Taking into account the required sample frequency in such experiments carried out with E. coli, which is in the order of seconds to sub-seconds (Schaefer et al, 1999;Buziol et al, 2002), the withdrawal of two samples for each single measurement is clearly not feasible. The question is, however, how fast the expected changes are in the levels of the metabolites which are present in the culture supernatant.…”

“…To achieve shorter intervals between samples Schaefer et al, (1999) constructed an automated sampling device with a maximum sampling frequency of 4.5 samples.s -1 . Another sampling device, which was based on the stopped-flow technique, was introduced by Buziol et al, 2002 with which dynamic responses in the millisecond range could be captured. Recently Schaub et al, 2006 proposed an integrated sampling procedure; whereby the sampling is carried out through a helical single tube heat exchanger.…”

Reconstruction of the O2 uptake rate and CO2 evolution rate on a time scale of seconds

“…In previous pulse-response studies the responses of gaseous compounds Ž . such as oxygen and carbon dioxide have not been measured ŽBuchholz et al, 2002;Buziol et al, 2002;Rizzi et al, 1997;Schmitz et al, 2002;Theobald et al, 1997;Vaseghi et al, . 1999;Visser et al, 2000 .…”

Global metabolite analysis of yeast: evaluation of sample preparation methods

“…These methods have been widely used in the extraction of metabolites from animal and plant tissues (Shryock et al, 1986;Kopka et al, 1995;Chen et al, 1998;Adams et al, 1999;Bouchereau et al, 2000;Conneely et al, 2002) and microorganisms (Cordeiro and Freire, 1996;Weuster-Botz, 1997;Hajjaj et al, 1998;Schaefer et al, 1999;Vaseghi et al, 1999;Letisse and Lindley, 2000;Martins et al, 2001;Buchholz et al, 2002;Buziol et al, 2002;Maharjan and Ferenci, 2003). Acidic extraction is commonly used for the extraction of nucleotides and water-soluble metabolites (Vaseghi et al, 1999;Martins et al, 2001;Buchholz et al, 2002), but for metabolome studies it is evident that not all metabolites are stable at the extreme pH, and destruction of some nucleotides has been reported during acidic extractions (Hajjaj et al, 1998).…”