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

Abstract

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.

The use of confocal scanning laser microscopy and other tools to characterize Escherichia coli in a high‐cell‐density synthetic biofilm

“…In addition, the standard film thicknesses were significantly smaller than the 50-300-pm active region determined for many aerobic systems (Bungay et al, 1969;Castillo et al, 1991;Chang and Moo-Young, 1988;Kurosawa et al, 1989;Monbouquette et al, 1990;Muller et al, 1994). .These layer thicknesses were within the range where actual thickness correlated with the theoretical values provided by R. D. Specialties.…”

“…For example, in aerobic sys-tems, only those cells contained in a thin layer that is close to the oxygen and substrate source are actually active (Bungay et al, 1969;Chang and Moo-Young, 1988;Karel and Robertson, 1989a;Kurosawa et al, 1989;Muller et al, 1994). However, both experimental and theoretical research weakens this argument, because results indicate that when cells are immobilized uniformly throughout a support, much of the biocatalyst is inactive.…”

Gel Entrapment and Micro-Encapsulation: Methods, Applications and Engineering Principles

“…Based on these observations, it was proposed that the immobilisation may have altered the normal cell cycle by preventing bud development in some cells undergoing DNA replication and nuclear division. Monbouquette et al, 1990;Kühn et al, 1991;Ollis, 1995Karel et al, 1987Stewart and Robertson, 1988;Karel and Robertson, 1989a, b de Beer et al, 1990;Geesey and White, 1990;Hooijmans et al, 1990a, b, c;Revsbech et al, 1989;Cronenberg et al, 1991Cronenberg et al, , 1994Massen et al, 1994;Müller et al, 1994;Beulingetal., 1995;Van den Heuvel, 1995 Willaertetal., 1990;Willaert, 1993Willaert, :1995 e.g. Eikmeier et al, 1984;Bailliez et al, 1985;Godia et al, 1987;Marin-Iniestaet al, 1988;Barbotin et al, 1990b Willaert and Baron, 1993 growth rate, spatial invasive biomass distribution Membrane reactor Measurements of On-line and in situ substrate and measurement product Hannoun and Stephanopoulos, 1990;Willaertetal., 1990;De Backer et al, 1992 Fluorescent measurements of intracellular NADH of S. cerevisiae cells grown on a gelatine surface and free cells have been performed (Doran and Bailey, 1987): dynamic responses of NADH for immobilised cells were different from those of yeast cells grown in suspension, indicating that immobilisation affects cellular regulation and control functions within the glycolytic pathway.…”

The use of confocal scanning laser microscopy and other tools to characterize Escherichia coli in a high-cell-density synthetic biofilm

“….These layer thicknesses were within the range where actual thickness correlated with the theoretical values provided by R. D. Specialties. In addition, the standard film thicknesses were significantly smaller than the 50-300-pm active region determined for many aerobic systems (Bungay et al, 1969;Castillo et al, 1991;Chang and Moo-Young, 1988;Kurosawa et al, 1989;Monbouquette et al, 1990;Muller et al, 1994). The films in this work were purposely designed to be much thinner than what would appear necessary based on these published results.…”

“…However, both experimental and theoretical research weakens this argument, because results indicate that when cells are immobilized uniformly throughout a support, much of the biocatalyst is inactive. For example, in aerobic sys-tems, only those cells contained in a thin layer that is close to the oxygen and substrate source are actually active (Bungay et al, 1969;Chang and Moo-Young, 1988;Karel and Robertson, 1989a;Kurosawa et al, 1989;Muller et al, 1994). This active region varies from approximately 30 p m thick in reactors with high cell density, such as that obtained in a hollow fiber reactor (Karel and Robertson, 1989b), to as thick as 300 p m in other systems (Kurosawa et al, 1989).…”