Hemicellulose residues can be hydrolyzed into a sugar syrup using dilute mineral acids. Although this syrup represents a potential feedstock for biofuel production, toxic compounds generated during hydrolysis limit microbial metabolism. Escherichia coli LY01, an ethanologenic biocatalyst engineered to ferment the mixed sugars in hemicellulose syrups, has been tested for resistance to selected organic acids that are present in hemicellulose hydrolysates. Compounds tested include aromatic acids derived from lignin (ferulic, gallic, 4-hydroxybenzoic, syringic, and vanillic acids), acetic acid from the hydrolysis of acetylxylan, and others derived from sugar destruction (furoic, formic, levulinic, and caproic acids). Toxicity was related to hydrophobicity. Combinations of acids were roughly additive as inhibitors of cell growth. When tested at concentrations that inhibited growth by 80%, none appeared to strongly inhibit glycolysis and energy generation, or to disrupt membrane integrity. Toxicity was not markedly affected by inoculum size or incubation temperature. The toxicity of all acids except gallic acid was reduced by an increase in initial pH (from pH 6.0 to pH 7.0 to pH 8.0). Together, these results a re consistent with the hypothesis that both aliphatic and mononuclear organic acids inhibit growth and ethanol production in LY01 by collapsing ion gradients and increasing internal anion concentrations.