KINETIC, DYNAMIC, AND PATHWAY STUDIES OF GLYCEROL METABOLISM BY KLEBSIELLA-PNEUMONIAE IN ANAEROBIC CONTINUOUS-CULTURE .2. ANALYSIS OF METABOLIC RATES AND PATHWAYS UNDER OSCILLATION AND STEADY-STATE CONDITIONS

The oscillation phenomena reported in the preceding article for the an aerobic continuous fermentation of glycerol by Klebsiella pneumoniae a re analyzed in terms of metabolic fluxes (metabolic rates and yields) and stoichiometry of pathways. Significant oscillations in the fluxes of CO2, H-2, formic acid, ethanol, and reducing equivalents are observ ed which show obvious relationships to each other. Changes in the cons umption or production rates of glycerol, acetic acid, 1,3-propanediol, and ATP are irregular and have relatively small amplitudes compared w ith their absolute values. By comparing the metabolic fluxes under osc illation and steady state that have nearly the same environmental cond itions it could be shown that pyruvate metabolism is the main step aff ected under oscillation conditions. The specific formation rates of al l the products originating from pyruvate metabolism (CO2, H-2, formic acid, ethanol, acetic acid, lactic acid, and 2,3-butanediol) show sign ificant differences under conditions of oscillation and steady state. In contrast, the specific rates of substrate uptake, ATP generation, a nd formation of products deriving either directly from glycerol (1,3-p ropanediol) or from the upstream of pyruvate metabolism (e.g., succini c acid) are not, or at least not significantly, affected during oscill ation. Stoichiometric analysis of metabolic pathways confirms that oth er enzyme systems, in addition to pyruvate: formate-lyase, must be sim ultaneously involved in the pyruvate decarboxylation under both oscill ation and steady-state conditions. The results strongly suggest oscill ations of activities of these enzymes under oscillation conditions. It appears that the reason for the occurrence of oscillation and hystere sis lies in an unstable regulation of pyruvate metabolism of different enzymes triggered by substrate excess and drastic change(s) of enviro nmental conditions. (C) 1996 John Wiley & Sons, Inc.