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The flask was incubated in a incubator shaker at 37° C. and 250 rpm overnight (about 16 hours). The entire flask contents then were used to inoculate the fermenter which was maintained at 37° C. The medium in the fermenter was aerated to allow fast growth of the organism. After six hours when the required cell mass was achieved, the following actions were taken: 1. Air was turned off to exert anaerobic conditions, which would initiate production of succinic acid; 2. Carbon dioxide gas was sparged into the medium at a rate of 0.03 mL per minute; and 3. A feed solution which contained the Arkenol's hydrolysate diluted with deionized water to a concentration of 500 g/L of total glucose plus xylose was added to the fermenter to achieve a total sugar concentration of 50 g/L in the fermentation medium. During the course of the experiment, when the sugar concentration in the fermnetr was low, more feed was added to provide sufficient substrates for succinic acid(cas:110-15-6) production. As the cells produced succinic acid(cas:110-15-6) the pH dropped. It was maintained at pH 6.5 by addition of a 1.5 M Na2CO3 solution through the action of an automatic pH controller. Samples were taken at intervals and analyzed for optical density, glucose, xylose, succinic acid(cas:110-15-6), acetic acid, lactic acid, and ethanol.
During the course of the experiment, when the sugar concentration in the fermenter was low, more feed was added to provide sufficient substrates for succinic acid(cas:110-15-6) production. As the cells produced succinic acid(cas:110-15-6), the pH dropped. It was maintained at pH 6.5 by addition of a 1.5 M Na2CO3 solution through the action of an automatic pH controller. Samples were taken at intervals and analyzed for optical density, glucose, xylose, succinic acid(cas:110-15-6), acetic acid, lactic acid, and ethanol.
Table 3, infra, and FIG. 3 illustrate the succinic acid(cas:110-15-6) production resulting from the utilization of the synthetic sugar mixture.
As can be noted in a comparison between Example 1 and Example 2, succinate production of the mutant was equivalent (see time points 120 and 122 of Table 2 and 3, respectively) when industrial hydrolysate was used versus when the synthetic feedstock was used. This result illustrates the robust character of the invented protocol in that any toxic materials inherent with industrial grade hydrolysates did not degrade the yield.
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