Shake-flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10-fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.