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The ability of acetaldehyde (90 mg l(-1)) to stimulate ethanol-stressed S. cerevisiae fermentations is examined and reasons for the effect explored. Alternative metabolic electron acceptors generated similar stimulatory effects to acetaldehyde, decreasing the ethanol-induced growth lag phase from 9 h to 3 h, suggesting a redox-driven effect. The exposure to ethanol caused an instant 60% decline in intracellular NAD(+) which was largely prevented by the addition of acetaldehyde. Furthermore, the exposure to ethanol affected glycolysis by decreasing the rate of glucose utilisation from 0.33 g glucose g(-1) biomass h(-1) to 0.11 g glucose g(-1) biomass h(-1), while the addition of acetaldehyde to an ethanol stressed culture increased this rate to 0.14 g glucose g(-1) biomass h(-1).

Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded.

SUMMARY Estimates of intracellular ethanol concentration in batch culturesof Saooharomyoes cerevisiae were significantly affected by continued fermentation during processing of the sample. Precooling of samples to 4°C and reduction of centrifugation time significantly reduced apparent intracellular ethanol concentration.Intracellular ethanol concentrations were substantially lower than in some previous reports and fell below the extracellular ethanol concentrations during the later stages of fermentation. I NTRODUCTI ONA number of recent reports suggest that the concentration of ethanol within fermenting yeast cells may be many times higher than the extracellular ethanol concentration