On-line stoichiometry and identification of metabolic state under dynamic process conditions

A method for the on-line calculation of conversion rates and yield coeffici ents under dynamic process conditions was developed. The method is based on cumulated mass balances using a moving average method. Elemental balances were used to test the measured cumulated quantities for gross errors and in appropriate stoichiometry definition followed by data reconciliation and es timation of non-measured conversion rates, using a bioprocess set-up includ ing multiple on-line analysis techniques. The quantitative potential of the proposed method is demonstrated by executing transient experiments in aero bic cultures of Saccharomyces cerevisiae on glucose. Rates and yield coeffi cients could be consistently quantified in shift-up, shift-down, and accele rostat experiments. The method shows the capability to describe quantitativ ely transient changes in metabolism including uncoupling of catabolism and anabolism, also for the case when multiple components of metabolism are not measured. The validity of the experiment can be evaluated on-line. Additio nally, the method detects with high sensitivity inappropriate stoichiometry definition, such as a change in state of metabolism. It was shown that con centration values can be misleading for the identification of the metabolic state. In contrast, the proposed method provides a clear picture of the me tabolic state and new physiological regulations could be revealed. Hence, t he novelty of the proposed method is the on-line availability of consistent stoichiometric coefficients allowing a significant speed up in strain char acterization and bioprocess development using minimal knowledge of the meta bolism. Additionally, it opens up the use of transient experiments for phys iological studies. (C) 2001 John Wiley & Sons, Inc.