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Metabolites 2016, 6(2), 12; doi:10.3390/metabo6020012

Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae

Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands
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Academic Editor: Peter Meikle
Received: 29 February 2016 / Revised: 13 April 2016 / Accepted: 20 April 2016 / Published: 23 April 2016
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Abstract

Ammonium (NH4+) is the most common N-source for yeast fermentations, and N-limitation is frequently applied to reduce growth and increase product yields. While there is significant molecular knowledge on NH4+ transport and assimilation, there have been few attempts to measure the in vivo concentration of this metabolite. In this article, we present a sensitive and accurate analytical method to quantify the in vivo intracellular ammonium concentration in Saccharomyces cerevisiae based on standard rapid sampling and metabolomics techniques. The method validation experiments required the development of a proper sample processing protocol to minimize ammonium production/consumption during biomass extraction by assessing the impact of amino acid degradation—an element that is often overlooked. The resulting cold chloroform metabolite extraction method, together with quantification using ultra high performance liquid chromatography-isotope dilution mass spectrometry (UHPLC-IDMS), was not only more sensitive than most of the existing methods but also more accurate than methods that use electrodes, enzymatic reactions, or boiling water or boiling ethanol biomass extraction because it minimized ammonium consumption/production during sampling processing and interference from other metabolites in the quantification of intracellular ammonium. Finally, our validation experiments showed that other metabolites such as pyruvate or 2-oxoglutarate (αKG) need to be extracted with cold chloroform to avoid measurements being biased by the degradation of other metabolites (e.g., amino acids). View Full-Text
Keywords: intracellular ammonium; UHPLC-IDMS; metabolomics; in vivo quantification; rapid sampling intracellular ammonium; UHPLC-IDMS; metabolomics; in vivo quantification; rapid sampling
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Cueto-Rojas, H.F.; Maleki Seifar, R.; ten Pierick, A.; Heijnen, S.J.; Wahl, A. Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae. Metabolites 2016, 6, 12.

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