Zymomonas mobilis ATCC 53431, a fructose utilization negative mutant, was found to be fructokinase negative and was optimized for a simultaneous production of fructose plus ethanol, minimizing sorbitol formation.
This mutant strain exhibits excellent growth at an initial pH of 6.5 and 7.0. However, the good growth does not correlate well with its fermentation activity.
With an initial pH set at 6.5 ethanol production prefers a pH control in the more acidic range between 4.5 and 5.0 whereas fructose formation becomes optimal in the more neutral range between 5.7 and 6.0. The formation of sorbitol as an undesirable by-product was found to be inversely related to the pH control, which means a stronger formation occurred at lower pH control values. Since a pH control above 6.0 clearly retarded growth and fermentation, most of the experiments were initiated at a pH of 6.5 and controlled at 6.0.
Temperature in the range of 25-35°C does not show any obvious effect on sucrose hydrolysis and fermentation using the continuous culture technique, but there was a trend towards lower biomass formation at the higher temperatures. An increase of temperature above 35°C was detrimental for both growth and fermentation of the mutant strain.
The addition of 30 mg/l ferrous sulfate, 0.02mM (2.96 mg/l) of cobalt chloride and 20 mg/l of Tween 80 to the maintenance medium improved significantly fructose formation in reducing sorbitol levels although glucose accumulation and sucrose hydrolysis were slightly retarded. However, increasing the sucrose concentrations up to 30% (w/v) improved sucrose hydrolysis efficiency to 98%. These results indicated that the added minerals must affect the glucose uptake mechanism.
Preculturing the inoculum in glucose rather than sucrose containing media improved glucose uptake and resulted in higher ethanol yields and fructose recovery, but did not avoid high glucose accumulation.
At 0.05 h-1 and 25% (w/v) sucrose in the feed stream, continuous culture resulted in a maximal fructose concentration of 89.09 g/l and a very much improved fructose:sorbitol ratio of 30.1. However, no true steady state could be obtained with the mutant strain.
It was the fed-batch culture finally, which gave the required results. Using the optimal conditions and sucrose concentrations of 20-25% (w/v) with a feeding rate of 50 ml/h, fructose concentrations reached 90 g/l with a fructose:sorbitol ratio between 20 and 25. This cultivation technique resulted in an approximately 90% pure fructose concentration in the medium with negligible residues of sucrose, glucose and sorbitol.
During these experiments it was observed that despite a near 98% sucrose hydrolysis, fructose formation did not reach the equivalent theoretical yield, which did improve considering the traces of sorbitol being produced. The question arose whether a leakage occurs with some fructose being metabolized to ethanol indicating that the fructose utilization negative mutant may not be a fructokinase negative mutant.
Enymatic investigations gave evidence that the mutant is a fructokinase negative mutant. A comparison between the glucokinases from the mutant and the wild strain ATCC 29191 indicated similar kinetic characteristics, identical electrophoretic mobility and molecular weight of 96,000 was found in the mutant's glucokinase. Both glucokinases, however, were able to phosphorylate fructose at concentrations in excess of 20 mM with an apparent Km for fructose by glucokinase of 177.94 and 177.07 mM respectively.
This discovery of fructose phosphorylation by the enzyme glucokinase not only explained the earlier observed leakage, but also indicates a separate regulatory mechanism of fructose transport and fructokinase.