The Gram negative bacterium E. coli is used in a wide range of biotechnological applications, as a result of straight forward cloning strategies and easy available cultivation techniques. Recombinant proteins are often expressed as inclusion bodies (IB), which are agglomerations of mostly misfolded protein, or as soluble cytoplasmic products, which can also be translocated to the periplasm or even the extracellular space. While recovery of extracellular proteins is rather simple, intracellular proteins and inclusion bodies in particular, demand high complex down-stream processes, often involving very cost-intensive refolding and chromatographic steps. Strategies to reduce these downstream operations, for example genetic modifications in the recombinant target protein to change the transport to different compartments within the cell or reduce and even prevent a high abundance of the formation of IB, are of high interest. Beside those genetic modifications, strategies in controlling process parameters during the fermentation process came recently into the focus of research. Different feeding strategies using mixed feeds, temperature and dO2 control can often be used to strongly influence the bacterial cell in production of the target protein. In this study we investigated the release of a periplasmic protein - alkaline phosophatase (AP) - to the supernatant during a fermentation process using a mixed feed strategy. As a first step the viability of the cell had to be monitored during the process. Avoiding complete cell lysis is of high importance to prevent product degradation of even product loss during the cultivation. Associated lysis monitoring was performed at-line via flow cytometric analysis using different dyes for differentiating between dead and living cells. In the course of this study a new method for online measurement of viable cell concentration using electrochemical impedance spectroscopy was tested, and applied during batch and fedbatch phases. With the possibility to determine the viable cell concentration (VCC) at-line and online, different outer membrane permeabilizing agents in combination with heat shock experiments were used to increase the release of the periplasmic protein into the supernatant. Permeability of the cells was investigated using photometric assays for alkaline phosphatase as a periplasmic protein.