Cell mass synthesis and degradation by immobilized Escherichia coli

Abstract

Escherichia coli K-12 cells were grown in a confined volume using microporous hollow fiber membranes. The local cell concentrations in the reactors were above 400 g dry mass/L, in excess of the predicted limit based on the specific volume of free cells determined by tracer exclusion. Cell mass synthesis and degradation rates in these reactors were measured using radioisotope labeling with (35)S. Net accumulation of cell material persisted at these high cell densities. The rates of substrate uptake and cell growth were predicted from the theory of reaction and diffusion assuming that kinetics of cell metabolism are identical for free-living and immobilized cells. This theory was tested by comparison of overall rates and by the size of the region in which cell growth occurred, measured by autoradiography. A yield coefficient of 4 +/- 1 mol sulfur/mol glucose was measured, in agreement with the value determined for free-living cells in similar conditions. Cell growth occurs in a thin layer (10-30 microm), at a rate similar to the growth rate for free cells. Volume expansion by the cells as a consequence of proliferation induces convection of cell mass out of the growth region into a region of the reactor filled with starving cells, which then accumulate in the reactor. The combination of mass-balance and spatial distribution measurements made possible by the use of radioisotope labeling enables a direct test for mass transfer limitations, the determination of the intrinsic cell kinetics, and noninvasive measurements of cell growth in immobilized cell reactors.

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

“…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). Theoretical efforts demonstrate similar results.…”

Engineering Cellular Photocomposite Materials Using Convective Assembly

“…Cell-laden alginate gels [57,86] and hydrogels [87,88,89,90,91,92,93,94] are other possible forms of cellular composite biomimetic leaf materials. Researchers at The National Renewable Energy Laboratory have fabricated photoreactive Ca 2+ -alginate films containing Chlamydomonas reinhardtii (a microalgae) that evolve hydrogen for over 150 hours [57], but these coatings lack the thinness, adhesion and organization of convectively-assembled coatings.…”

In SituNMR Systems

“…The thickness of the growing region can be estimated by a reaction-diffusion model. A reactiondiffusion model was formulated for E. coli cells grown anaerobically with glucose as the limiting nutrient in a hollow fiber reactor (43,47). 31 P NMR results illustrate the concepts from this model quite elegantly.…”

“…Hollow fibers reactors for use in NMR experiments are commonly employed with cells confined in the shell side of the reactor and with nutrients being supplied by diffusion through the fiber wall (42)(43)(44)(45)(46)(47)(48). Membrane reactors can be diffusionally supplied (27,45) or convectively supplied (47), with more severe mass-transfer restrictions in the diffusionally supplied membrane reactor.…”