Toxic products can generally be divided into three main groups — aliphatic acids, furan de­rivatives and phenolic compounds [68—70] released by degradation of carbohydrates, and compounds arising from lignin. In acidic solutions, cellulose and hemicellulose are broken down into hexose and pentose sugars, which are further decomposed at high temperatures into furan derivatives represented mainly by 2-furaldehyde (furfural, FF) and 5-hydroxy- methyl-2-furaldehyde (hydroxymethylfurfural, HMF). Free aliphatic acids, represented mainly by acetic, formic or levulinic acids, are created by substituents cleaved from lignin and hemicelluloses within the pretreatment, or are produced by cells during fermentation, while phenolic derivatives (4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid or vanilin) arise mainly from lignin decomposition in alkaline solution [71]. About 40 lignocellulose degradation products have been identified in various hydrolysates [71], the type and amount depending on type of biomass and pretreatment conditions [68]; e. g. furfural, hy — droxymethylfurfural and levulinic acid occur in higher concentrations at low pH combined with high temperature and pressure [68, 71], while vanilin, vanilic, benzoic and 4-hydroxy — coumaric acids are formed under alkaline conditions at elevated temperatures and acetic acid is produced in significant concentrations independent of the process and type of bio­mass [71]. Although many studies on the effect of inhibitors on cellulolytic enzymes have been published, a general conclusion is not easy to draw because it is influenced not only by the type and origin of the enzyme preparation, but also by its dosing and the concentration of inhibitors. However, in general, compounds exhibiting higher hydrophobicity tend to be more inhibitory to cellulolytic enzymes, the greatest inhibitory effect being caused by acetic and formic acids [72—74], while the activity of enzymes is not practically influenced by levu — linic acid [73]. On the other hand, the presence of inhibitory compounds also affects ethanol productivity in the subsequent fermentation by influencing metabolic functions of ethanol producing strains. Inhibitory effects are described by type and concentration of toxic com­pounds (their effect is intensified when present in combination) and the strain used for etha­nol production, but generally, fermentation is mainly influenced by the presence of furan derivatives together with phenolic compounds and weak acids (at low pH). As reviewed elsewhere [70, 75], low molecular weight compounds are able to penetrate the cell, while in­hibitors with high molecular weights affect expression and activity of sugar and ion trans­porters. Growth and rate of ethanol production by Saccharomyces cerevisiae, the main ethanol producing strain, is significantly inhibited by furfural, while ethanol yield is almost not in­fluenced [75] due to its ability to detoxify the broth by reduction of furfural to furfuryl alco­hol, which is less toxic.

Surprisingly, in butanol production process, C. beijerinckii BA101, C. acetobutylicum P260, C. acetobutylicum ATCC 824, Clostridium saccharobutylicum 262 and Clostridium butylicum 592 were not sensitive towards sugar degradation products like furfural or hydroxymethylfur — fural (up to concentrations of 2-3 g/l) but its growth and solvent production were inhibited by p-coumaric and ferulic acids present at a concentration of 0.3 g/l [76—78]. Solvent produc­tivity and final solvent concentration in C. beijerinckii P260 were stimulated by addition of furfural or hydroxy methylfurfural (or both compounds) to the fermentation medium, at concentrations of up to 1 g/l [79]. C. acetobutylicum ATCC 824 metabolized furfural and hy­droxymethyl furfural into furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively and these compounds positively influenced solvent production up to a concentration of 2 g/l. It was hypothesised that this biotransformation step, independent of initial furfural and HMF concentrations, might increase solventogenesis via an increased rate of regeneration of NAD+ [80]. Another possible inhibitor of phenolic origin, syringaldehyde, caused inhibition of solvent production by C. beijerinckii NCIMB 8052 over the whole range tested (0.2-1 g/l). This inhibition was probably caused by decreased expression and activity of coenzyme A transferase, which participated in utilization of butyric and acetic acids, because these acids accumulated in the medium [81].

The inhibitory effects of toxic compounds released by sugars and lignin degradation can be reduced in several ways, e. g. optimization of pretreatment conditions to minimize the formation of inhibitors, use of specific detoxification methods, e. g. precipitation by calci­um hydroxide (overliming) alone or in combination with sulphite addition, adsorption on charcoal, evaporation of the volatile fraction, extraction with ethyl acetate or diethyl ether, ion extraction, treatment with peroxidase (E. C. 1.11.7) and laccase (EC 1.10.3.2), or use of microbial strains with increased resistance to inhibitors (achieved by adaptation or prepared by genetic modification) [75, 82, 83]. Lignin degradation products, p-coumaric, ferulic and vanillic acids, together with vanillin, were effectively removed from a model solution of phenolic compounds by treatment with 0.01|oM peroxidase (E. C. 1.11.7), re­sulting in improved growth and butanol production by C. beijerinckii NCIMB 8052 [84]. Sulphuric acid-hydrolysed corn fiber was treated with XAD-4 resin, resulting in an im­provement of butanol yield achieved with C. beijerinckii BA101 [85]. Another popular ap­proach for detoxification of acid hydrolysates for butanol production is "overliming" i. e. addition of Ca(OH)2 in excess to hydrolysate [78, 85]. Although this detoxification meth­od has been known for a long time, its mode of action, especially in the case of butanol production, is not completely clear. Addition of Ca(OH)2 to an acid hydrolysate decreases furfural and HMF concentrations [86, 87] but does not affect acid concentrations; thus it is only possible to assume a beneficial neutralization effect. Furthermore it may be useful to treat hydrolysates with activated carbon [88].