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Four strains of the homofermentative, obligately anaerobic thermophile Clostridium thermoaceticum were compared in pH-controlled batch fermentation for their tolerance to acetic acid, efficiency of converting glucose to acetic acid and cell mass, and growth rate. At pH 6 (and pH 7) and initial acetic acid concentrations of less than 10 g/liter, the four strains had mass doubling times of 5 to 7 h and conversion efficiencies to acetic acid and cell mass of about 90% (70 to 110%) and 10%, respectively. At pH 6 and initial acetic acid concentrations of greater than 10 g/liter, only two of the strains grew, the mass doubling time increased to 18 h, and the conversion efficiencies to acetic acid and cell mass remained unchanged. Both of these strains had been selected for their ability to grow in the presence of acetate at neutral pH. The highest acetic acid concentrations reached were about 15 and 20 g/liter at pH 6 and 7, respectively. C. thermoaceticum is apparently more sensitive to free acetic acid than to either acetate ion or pH. It was also shown that, at pH 6 and 7, the redox potential must be at least as low as −300 and −360 mV, respectively, for growth to occur.

Using a series of pH controlled batch fermentations operated in a fed-batch mode and adaptation and selection techniques where pH and acetic acid provided the selective pressures, we isolated a culture of Clostridium thermoaceticum that can grow and produce acetic acid at pH 4.5. At pH 4.5 the fastest mass doubling time was 36 h, and the highest acetic acid concentration reached was 4.5 g/liter. Generally, as the pH was decreased from 6.0 and the initial acetic acid concentration increased, the mass doubling time increased, and the final acetic acid concentration decreased. These observations can be explained in terms of inhibition by the free acetic acid concentration at a given pH, relative to the total acetic acid concentration (free acid plus acetate ion). We have thus reached one of the criteria determined by us to be required for an economically viable fermentation acetic acid process, i.e., pH 4.5. A second requirement for a mass doubling time of about 7 h (0.1/h dilution rate) can probably be reached by selection in continuous culture. The final requirement for an acetic acid concentration of 50 g/liter will be the most difficult to achieve in view of the organism's sensitivity to low concentrations of free acetic acid.

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