Real and pseudo oxygen gradients in Ca-alginate beads monitored during polarographic Po2-measurements using Pt-needle...

Polarographic microcoaxial needle electrodes were used to measure internal profiles of dissolved oxygen tension (Po(2)) within single Ca-alginate beads of different diameter containing entrapped cells of Saccharomyces cerevisiae. For the investigations, single beads coming from variable growing conditions and distinct cultivation stages were fixed in a special holding device. In dependence on microbial growth steep oxygen gradients were observed. The Oxygen penetration depth at steady state lay between 50 and 100 microm. After 8 h of cultivation time, the anaerobic space within the beads (phi 2 mm; cultivation in a packed bed reactor) is beginning at approximately 130 microm, whereas the anaerobic space within the beads (phi 2 mm) coming from the shaker flask culture is located approximately 440 microm below the bead surface. Surprisingly, steep gradients were also observed, when recording profiles from cell-free Ca-alginate beads of different diameter and alginate concentrations. The steep oxygen gradients apparently had to be interpreted as pseudo-Po(2)-gradients. These results were borne by several effects, such as formation of artifacts and diffusion barriers in front of the electrode tip or oxygen "availability" at the tip and consumption of oxygen by the electrode itself. These phenomena could be documented by microscopic observation and photography. Thus, to obtain real Po(2)-profiles it is important to be exactly informed about the physical, chemical, and biological properties of the material to be investigated. Furthermore, it is necessary to apply a special stepwise puncture technique with distinct step-in/step-out movements of the electrode: e.g., unidirectional or contradirectional puncture techniques.