A number of components in lignocellulosic hydrolysates can inhibit the growth and ethanol production of bacteria and yeasts, and acetic acid has been identified as a major potential inhibitor of Z. mobilis in such acid — produced hydrolysates [61-65]. Lawford and Rousseau [32] examined the role of glucose feeding as a means of improving fermentation perform­ance in acetate-containing media. Another approach to solving this problem has been to use a hydrolysate-fed chemostat to produce adapted or mutant strains [33,34]. Following chemical mutagenesis, Joachimsthal et al. [66] iso­lated a mutant strain, designated ZM4/AcR with a higher acetate resistance than the parent strain. This strain was then transformed by Jeon et al. [67] to the mutant recombinant ZM4/AcR (pZB5). Compared to ZM4 (pZB5), this strain showed enhanced kinetics in batch culture in the presence of 12 gL-1 sodium acetate (8.8 g L-1 acetic acid) at pH = 5.0 in batch culture on 40 g L-1 glucose, 40 g L-1 xylose medium. In continuous culture there was evidence of increased maintenance energy requirements/uncoupling of metabolism in the presence of acetate.

In more recent studies Saez-Miranda et al. [68] have determined ATP levels for growth on glucose/xylose media in the presence of different concentra­tions of acetic acid. From their results they have found that ATP production and accumulation rates are most sensitive to acetic acid at lower pH values— a result consistent with the earlier NMR studies by Kim et al. [57] which demonstrated increasing de-energization of the cells as the inhibitory effects of acetic acid increased. The greater toxicity of acetic acid at lower pH is related to its pKa value as only unprotonated acid can be transported into the cells.

The effects of a range of inhibitory compounds at levels reported previ­ously for a pre-treated hardwood hydrolysate [65] on specific rates of xylose utilization and ethanol production for ZM4 (pZB5) have been analyzed by Kim et al. [57]. From the results, sodium acetate was found to have the greatest inhibitory effect at the concentration tested (10.9 gL-1 at pH = 6.0), followed by vanillin (0.04 gL-1), syringaldehyde (0.13 gL-1) hydroxymethyl- furfural (0.9gL-1) and furfural (0.3 gL-1). Vanillic acid (0.08 gL-1) did not show any inhibitory effects at this experimental concentration. At the levels tested, these inhibitory compounds did not affect ethanol yields on xylose. Volumetric rates of xylose utilization and ethanol produc­tion were reduced by up to 20% by addition of the individual inhibitory components.

2.7