The fed-batch fermentation of glycerol to 1,3-propanediol by Klebsiella pneumoniae displayed an unusual dynamic behavior that can be clearly divided into four distinct phases according to cell growth and CO, evolution rate. Metabolism changed significantly during the different phases as reflected by the varied specific rates of substrate consumption and product formation. An assay of activities of the three initial enzymes of glycerol metabolism, namely glycerol dehydratase (GDHt), glycerol dehydrogenase (GDH), and 1,3-propanediol-oxidoreductase (PDOR), showed apparently different patterns of expression. To understand the culture dynamics and patterns of enzyme formation at a more systemic level we analyzed the expression patterns of intracellular proteins of K. pneumoniae from different phases of the fed-batch fermentation using two-dimensional gel electrophoresis (2DE). Two new enzymes, namely a phosphoenolpyruvate-dependent dihydroxyacetone kinase (DHAK II) and a hypothetical oxidoreductase (HOR), which are directly related to glycerol metabolism and 1,3-propanediol formation, were identified among the highly expressed proteins. The changes in expression of these new enzymes and several other proteins identified from the 2DE analysis helped to understand not only the dynamic behavior of the fed-batch fermentation reported in this work but also some previously insufficiently understood phenomena related to this fermentation process. In particular, we demonstrated the combined use of proteomic analysis and enzyme activity assay data for metabolic pathway analysis and for a better identification of targets for bioprocess improvement. (C) 2003 Wiley Periodicals, Inc.