Figure 2.

Culture heterogeneity of GFP producing B. megaterium cells (A and B). B. megaterium cells were cultivated on A5 medium¹⁹ agarose pad at 37°C and observed using a Zeiss Axiovert 200 M microscope. Pictures were taken using an AxioCam HR under 630x total magnification (63x objective, 10x ocular). (A) fluorescent image of GFP producing cells; (B) bright image of the same cells overlaid with green-colored fluorescent image. (C) A Biostat B2 bioreactor (B. Braun, Melsungen, Germany) with 2 L working volume connected to an exhaust gas analysis unit (Maihak, Hamburg, Germany) was operated and controlled as described previously.¹⁶,¹⁹ B. megaterium carrying a plasmid coding for GFP-Strep fusion protein was grown in semi-defined minimal medium at 37°C initially in a batch phase with 4 g/L glucose. At the end of the batch phase an exponential feeding profile was started. GFP was visualized by a lamp emitting blue light and a yellow filter using a digital camera. (D) Results of flow cytometric analysis of bioreactor cultivation. Samples taken from bioreactor cultivation of B. megaterium carrying a plasmid coding for GFP before and 4.6 h after induction of the gfp gene expression were stained with propidium iodide (PI) and analyzed in a FACSCalibur (Benton Dickinson, Belgium): Living cells, no GFP: red; living cells, GFP: green; dead cells, no GFP: black; dead cells, GFP: cyan. Percentages of the subpopulation compared to all cells are given.