While hemicellulose represents a large potential biomass source that is not presently utilized, pretreatment is required for depolymerization of its sol­uble components. Many depolymerization techniques are available, but re­search in this laboratory has focused on hydrolysis with dilute mineral acid at modest temperatures [85,86]. Unfortunately, dilute acid hydrolysis produces toxins that negatively affect biocatalyst growth and metabolism (reviewed in [87]); many of these toxins are listed in Fig. 1. Recent work has focused on an increased understanding of the underlying mechanisms of toxicity and methods for toxicity quantification and reduction.

Furfural, a pentose sugar derivative, is present in hemicellulose hydrolysate at a concentration of 1-4 gL-1 [88] but can inhibit E. coli growth at con­centrations as low as 2.4 gL-1 [89,90]. While other aldehydes, such as 4- hydroxybenzaldehyde and syringaldehyde, are more toxic than furfural on a weight basis, the presence of furfural enhances the effect of other tox­ins [90]. Despite the observed toxicity, ethanologenic E. coli KO11 and LY01 and K. oxytoca P2 have demonstrated a native ability to transform furfural to furfuryl alcohol [91]; the size and substrate specificity of the LY01 furfural re­ductase suggests that it is a new type of alcohol-aldehyde oxidoreductase [92]. Strain LY01, which has higher ethanol tolerance than KO11, also has higher furfural tolerance: KO11 growth was completely inhibited by 3 gL-1 furfural but LY01 was not, although growth was reduced by more than 50% [90]. Con­trastingly, there is no difference in the syringaldehyde tolerance of the two strains [90].

The toxicity of representative alcohol, aldehyde, and acid components of hemicellulose hydrolysis were investigated and found to affect ethanologenic E. coli LY01 in various ways [90,93,94]. In all cases, toxicity was related to hy — drophobicity. The organic acid data suggests that aliphatic and mononuclear acids both inhibit biocatalyst growth and ethanol production by collapsing ion gradients and increasing the internal anion concentration, and not by in­hibiting central metabolic or energy pathways [93]. At least some inhibitors are present at sufficient concentrations to account for the observed growth in­hibition: 9 g L-1 of acetic acid in rich media inhibits LY01 growth by 50%, and acetic acid concentration in hydrolysate can exceed 10 gL-1.

While all of the tested aldehydes did inhibit growth, only furfural had an impact on ethanol production [90]. Alcohols have a lower toxicity than alde­hydes and acids and appeared to inhibit ethanol production primarily by inhibiting growth [94].

Total furan content is representative of total toxicity and can be estimated from UV spectra [95]. The adjustment of hydrolysate pH to 9-10 by the add­ition of Ca(OH)2, a process known as overliming, is an effective method of hydrolysate toxicity reduction [96]. LY01 was able to produce less than 1 g L-1 ethanol from hydrolysate adjusted only to pH 6.5-6.7 but produced 33 gL 1 ethanol from baggase hydrolysate that was overlimed to pH 11 [97].

4.4