Bioprocess and Biosystems Engineering

, Volume 32, Issue 5, pp 581–592 | Cite as

Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock

  • Max Wellerdiek
  • Dajana Winterhoff
  • Waldemar Reule
  • Jürgen Brandner
  • Marco Oldiges
Original Paper

Abstract

Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps.

Keywords

Methanol quenching Metabolome analysis Metabolomics Microstructure heat exchanger Rapid sampling 

Nomenclature:

G6P

Glucose 6-phosphate

F6P

Fructose 6-phosphate

E4P

Erythrose 4-phosphate

PYR

Pyruvate

DHAP

Dihydroxyacetone phosphate

GAP

Glyceraldehyde 3-phosphate

R5P

Ribose 5-phosphate

S7P

Seduheptulose 7-phosphate

NAD+

Nicotinamide adenine dinucleotide

NADH

Nicotinamide adenine dinucleotide (reduced form)

2/3PG

2-/3-Phosphoglycerate

ATP

Adenosinetriphosphate

ADP

Adenosinediphosphate

AMP

Adenosinemonophosphate

PEP

Phosphoenolpyruvate

FBP

Fructose 1,6-bisphosphate

CitICIT

Citrate/isocitrate

ACN

cis-Aconitate

NADP+

Nicotinamide adenine dinucleotide-phosphate

NADPH

Nicotinamide adenine dinucleotide-phosphate (reduced form)

Rib5P

Ribulose 5-phosphate

Notes

Acknowledgments

This project was financially supported by the German Ministry of Research and Education (BMBF, grant 0313703) and Evonik Degussa GmbH. The authors would like to thank Prof. Christian Wandrey for the excellent working conditions at the Institute of Biotechnology 2, Forschungszentrum Jülich GmbH.

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Max Wellerdiek
    • 1
  • Dajana Winterhoff
    • 1
  • Waldemar Reule
    • 3
  • Jürgen Brandner
    • 2
  • Marco Oldiges
    • 1
  1. 1.Institute of BiotechnologyForschungszentrum Jülich GmbHJülichGermany
  2. 2.Institute of Micro Process EngineeringForschungszentrum Karlsruhe GmbHEgenstein LeopoldshafenGermany
  3. 3.Hochschule FurthwangenFurtwangenGermany

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