Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose.

Escherichia coli FBR5 containing recombinant genes for ethanol production on plasmids that are also required for anaerobic growth was cultivated continuously on 50 g/l xylose or glucose in the absence of antibiotics and without the use of special measures to limit the entry of oxygen into the fermenter. Under chemostat conditions, stable ethanol yields of ca. 80-85% of the theoretical were obtained on both sugars over 26 days at dilution rates of 0.045/h (xylose) and 0.075/h (glucose), with average plasmid retention rates of 96% (xylose) and 97% (glucose). In a continuous fluidized bed fermenter, with the cells immobilized on porous glass beads, the extent of plasmid retention by the free cells fell rapidly, while that of the immobilized cells remained constant. This was shown to be due to diffusion of oxygen through the tubing used to recirculate the medium and free cells. A change to oxygen-impermeable tubing led to a stable high rate of plasmid retention (more than 96% of both the free and immobilized cells) with ethanol yields of ca. 80% on a 50 g/l xylose feed. The maximum permissible level of oxygen availability consistent with high plasmid retention by the strain appears to be of the order of 0.1 mmol per hour per gram dry biomass, based on measurements of the rate of oxygen penetration into the fermenters. Revertant colonies lacking the ethanologenic plasmid were easily detectable by their morphology which correlated well with their lack of ampicillin resistance upon transfer plating.