How do intracellular fluxes respond to dynamically increasing glucose limitation when the physiology changes from strong overflow metabolism near to exclusively maintenance metabolism? Here we investigate this question in a typical industrial, glucose-limited fed-batch cultivation with a riboflavin Overproducing Bacillus subtilis strain. To resolve dynamic flux changes, a novel approach to C-13 flux analysis was developed that is based on recording C-13 labeling patterns in free intracellular amino acids. Fluxes are then estimated with stationary flux ratio and iterative isotopomer balancing methods, for which a decomposition of the process into quasi-steady states and estimation of isotopic steady state C labeling patterns was necessary. By this approach, we achieve a temporal resolution of 30-60 min that allows us to resolve the slow metabolic transients that typically occur in Such cultivations. In the late process phase we found, most prominently, almost exclusive respiratory metabolism, significantly increased pentose phosphate pathway contribution and a strongly decreased futile cycle through the PEP carboxykinase. As a consequence, higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands, suggesting a transhydrogenase-like mechanism to close the balance of reducing equivalents. Biotechnol. Bioeng. 2010;105: 795-804. (C) 2009 Wiley Periodicals, Inc.