Conditions that promote production of lactic acid by Zymomonas mobilis in batch and continuous culture

This study documents the similar pH-dependent shift in pyruvate metabolism exhibited by Zymomonas mobilis ATCC 29191 and ATCC 39676 in response to controlled changes in their steady-state growth environment. The usual high degree of ethanol selectivity associated with glucose fermentation by Z. mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and CO2, and the remaining 5% being used for the synthesis of cell mass. Conditions that promote energetic uncoupling cause the conversion efficiency to increase to 98% as a result of the reduction in growth yield (cell mass production). Under conditions of glucose-limited growth in a chemostat, with the pH controlled at 6.0, the conversion efficiency was observed to decrease from 95% at a specific growth rate of 0.2/h to only 80% at 0.042/h. The decrease in ethanol yield was solely attributable to the pH-dependent shift in pyruvate metabolism, resulting in the production of lactic acid as a fermentation byproduct. At a dilution rate (D) of 0.042/h, decreasing from pH 6.0 to 5.5 resulted in a decrease in lactic acid from 10.8 to 7.5 g/L. Lactic acid synthesis depended on the presence of yeast extract (YE) or tryptone in the 5% (w/v) glucose-mineral salts medium. At D = 0.15/h, reduction in the level of YE from 3 to 1 g/L caused a threefold decrease in the steady-state concentration of lactic acid at pH 6. No lactic acid was produced with the same mineral salts medium, with ammonium chloride as the sole source of assimilable nitrogen. With the defined salts medium, the conversion efficiency was 98% of theoretical maximum. When chemostat cultures were used as seed for pH-stat batch fermentations, the amount of lactic acid produced correlated well with the activity of the chemostat culture; however, the mechanism of this prolonged induction effect is unknown. The levels of lactic acid produced by Z. mobilis in this study have not been previously reported. Zymomonas is Gram-negative, and at no time did microscopic inspection of lactic-acid-producing cultures indicate the presence of Gram-positive organisms. Although these observations are very preliminary in nature, they have implications for the regulation of glycolytic flux in Zymomonas, and demonstrate the possibility of an alternative fate for pyruvate previously presumed not to exist.