Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry.

Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria.

Optimization of antibody fragment production in Bacillus megaterium: the role of metal ions on protein secretion.

The effect of the concentration of metal ions in minimal media has been shown to be very important for the production and secretion of the antibody fragment D1.3 scFv in Bacillus megaterium YYBm1. The best media compositions for biomass and antibody fragment formation were evaluated using a genetic algorithm. The screening was carried out in 96 microtiter deep well plates with 900 µL cultivation volume. In 7 generations, 240 different kinds of media were tested, key elements for production and secretion were detected and a 117% increase in production of antibody fragment compared to the previously used medium could be achieved. In addition, media with a higher biomass formation (+84%) or with both more biomass and a higher production of antibody fragment (Pareto-front members) were found. Interestingly the best media for protein production and secretion were different in their composition, with regards to the metal ion concentration levels. From data derived experimentally and from the genome, magnesium was shown to be one of the key components of the metal ions tested for biomass formation and especially for production and secretion of the antibody fragment D1.3 scFv.

Process optimization of the integrated synthesis and secretion of ectoine and hydroxyectoine under hyper/hypo-osmotic stress.

The synthesis and secretion of the industrial relevant compatible solutes ectoine and hydroxyectoine using the halophile bacterium Chromohalobacter salexigens were studied and optimized. For this purpose, a cascade of two continuously operated bioreactors was used. In the first bioreactor, cells were grown under constant hyperosmotic conditions and thermal stress driving the cells to accumulate large amounts of ectoines. To enhance the overall productivity, high cell densities up to 61 g L(-1) were achieved using a cross-flow ultrafiltration connected to the first bioreactor. In the coupled second bioreactor the concentrated cell broth was subjected to an osmotic and thermal down-shock by addition of fresh distilled water. Under these conditions, the cells are forced to secrete the accumulated intracellular ectoines into the medium to avoid bursting. The cultivation conditions in the first bioreactor were optimized with respect to growth temperature and medium salinity to reach the highest synthesis (productivity); the second bioreactor was optimized using a multi-objective approach to attain maximal ectoine secretion with simultaneous minimization of cell death and product dilution caused by the osmotic and thermal down-shock. Depending on the cultivation conditions, intracellular ectoine and hydroxyectoine contents up to 540 and 400 mg per g cell dry weight, respectively, were attained. With a maximum specific growth rate of 0.3 h(-1) in defined medium, productivities of approximately 2.1 g L(-1) h(-1) secreted ectoines in continuous operation were reached.