Effects of Immobilization on the Metabolism of Yeast

Citation

Doran, Pauline Mavis (1985) Effects of Immobilization on the Metabolism of Yeast. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5445-rc32. https://resolver.caltech.edu/CaltechETD:etd-04032008-111952

Abstract

The composition and kinetic properties of Saccharomyces cerevisiae were found to be substantially different when the cells were immobilized on gelatin. Batch fermentation experiments conducted in a gradientless reaction system allowed comparison of immobilized cell and suspended cell performance.

In complete nutrient medium, the specific rate of ethanol production by the immobilized cells was 40-50% greater than for the suspended yeast. The immobilized cell consumed glucose twice as fast as the suspended cells, but their specific growth rate was reduced by 45%. Yields of biomass from the immobilized cell population were lower at one-third the value for the suspended cells.

Cellular composition was also affected by immobilization. Measurements of intracellular polysaccharide levels showed that the immobilized yeast stored larger quantities of reserve carbohydrates and contained more structural polysaccharide than suspended cells. Flow cytometry was used to obtain DNA, RNA and protein frequency functions for suspended and immobilized cell populations. These data showed that the immobilized cells have higher ploidy than cells in suspension. The level of stable, double-stranded RNA in immobilized cells was only one-quarter that measured for suspended cells. The observed changes in immobilized cell metabolism and composition may have arisen from disturbance to the yeast cell cycle by cell attachment, causing alterations in the normal patterns of yeast bud development, DNA replication and synthesis of cell wall components.

Recovery from hydroxyurea-induced DNA synthesis inhibition was indicated by measurements of growth rate, DNA content and light scatter properties. The immobilized cells quickly developed an effective means for overcoming hydroxyurea-induced inhibition of replication, and proceeded to synthesize large amounts of DNA while still in the presence of the inhibitor. Stable RNA levels for immobilized cells remained low at 25% of the measured quantity for hydroxyurea-treated suspended cells. Synthesis of protein and RNA was not adversely affected in either cell type. Suspended cell protein pools increased by a factor of 1.8 following inhibition, while the immobilized cells contained 2.6 times the level of protein before hydroxyurea treatment.

Fermentation properties of immobilized and suspended cells were changed by hydroxyurea. The specific rate of ethanol production by immobilized cells increased by an average of 24%, while, for the suspended cells, specific productivity was up to three times higher. Glucose consumption rates for both cell types also increased under the influence of hydroxyurea. Suspended cell polysaccharide content was reduced by 65%, while the immobilized cells, in contrast, contained 30% more polysaccharide after hydroxyurea treatment.

Biotin starvation of immobilized yeast was effective in reducing synthesis of DNA. Biotin-deficient immobilized S. cerevisiae contained approximately the same quantity of DNA as starved suspended cells, while RNA and protein levels were reduced. Glycerol was synthesized at the expense of ethanol during fermentation by biotin-deficient immobilized and suspended cells.

The character and occurrence of glycolytic oscillations were affected by immobilization. Endogenous metabolism by immobilized cells gave rise to relaxation oscillations in the absence of external substrate. Addition of glucose to starved immobilized yeast also generated relaxation-type behaviour, while suspended cells produced only sinusoidal waveforms. Interpretation of these immobilized cell dynamics is discussed in terms of the kinetics and regulatory properties of energy metabolism in yeast.

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