Recently, the production of various chemicals and fuels via microbial fermentation has gained momentum. Development of efficient bio-processes requires a system-scale understanding of the metabolic network of biocatalysts. In this context, we evaluated the roles of metabolic pathways and enzymes during fermentation of various substrates in E. coli using metabolic flux analysis (MFA), which is a powerful and efficient tool for comprehensive investigation of a biological system. The combination of substrates studied covered the full range of oxidation states of common carbon sources. During fermentation, pyruvate is a key precursor metabolite and a prominent intermediate for the synthesis of most fermentation products in E. coli. Under fermentative conditions, pyruvate is primarily dissimilated via pyruvate formate lyase (PFL), and pyruvate dehydrogenase (PDH) exhibits negligible activity with unknown physiological role. However, we found that the activity of PDH was required for efficient fermentative growth of E. coli. PDH was even able to support fermentative growth on glucuronate in a strain devoid of PFL. MFA indicated that a deletion of PDH leads to more than 10 fold increase in flux through oxidative pentose pathway. These results were supported by the 13C labeling based flux analysis. Subsequently, the hypothesized the role of PDH: to efficiently generate CO 2, assisting cell growth during glucose fermentation and to efficiently generate reducing equivalents aiding cell growth during glucuronate fermentation. In silico flux analysis was used to design further genetic modifications and supplementation experiments, which were instrumental in verifying our hypothesis. On the other hand, the fermentation of glycerol by E. coli, had been unknown until recently. In this study, we identified the factors facilitating this process in E. coli. Nuclear magnetic resonance (NMR) analysis of fermentation samples identified ethanol and 1,2-propanediol (1,2-PDO) as products of glycerol fermentation. Employing 13C tracer experiments, we demonstrated that majority of the fermentation products and about 20% of the biomass building blocks originate from glycerol. In silico flux analysis was instrumental in elucidating the role of active pathways during glycerol fermentation: redox-balanced pathway to ethanol generates energy for cell growth, and the redox consuming pathway synthesizing 1,2-PDO facilitates cell growth by enabling redox balance.