During hydrolysis of lignocellulosic biomass, monomeric sugars and a broad range of inhibitory compounds are formed and released. These inhibitors, which can be organized around three main groups, furans, weak acids and phenolics, reduce ethanol yield and productivity by affecting the microorganism performance during the fermentation step. Among the microorganisms that have been evaluated for lignocellulosic hydrolysate ethanol fermentation, the yeast Saccharomyces cerevisiae appears to be the least sensitive. In order to overcome the effect of inhibitors, strategies that include improvement of natural tolerance of microorganism and use of fermentation control strategies have been developed. An overview of the origin, effects and mechanisms of action of known inhibitors on S. cerevisiae is given. Fermentation control strategies as well as metabolic, genetic and evolutionary engineering strategies to obtain S. cerevisiae strains with improved tolerance are discussed.

The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cellfree crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity.

To meet the increasing need for bioenergy several raw materials have to be considered for the production of e.g. bioethanol and biogas. In this study, three lignocellulosic raw materials were studied, i.e. (1) winter rye straw (Secale cereale L), (2) oilseed rape straw (Brassica napus L.) and (3) faba bean straw (Viciafaba L.). Their composition with regard to cellulose, hemicellulose, lignin, extractives and ash was evaluated, as well as their potential as raw materials for ethanol and biogas production. The materials were pretreated by wet oxidation using parameters previously found to be optimal for pretreatment of corn stover (195 1C, 15 min, 2 g l À1 Na 2 CO 3 and 12 bar oxygen). It was shown that pretreatment was necessary for ethanol production from all raw materials and gave increased biogas yield from winter rye straw. Neither biogas productivity nor yield from oilseed rape straw or faba bean straw was significantly affected by pretreatment. Ethanol was produced by the yeast Saccharomyces cerevisiae during simultaneous enzymatic hydrolysis of the solid material after wet oxidation with yields of 66%, 70% and 52% of theoretical for winter rye, oilseed rape and faba bean straw, respectively. Methane was produced with yields of 0.36, 0.42 and 0.44 l g À1 volatile solids for winter rye, oilseed rape and faba bean straw, respectively, without pretreatment of the materials. However, biogas productivity was low and it took over 50 days to reach the final yield. It could be concluded that all three materials are possible raw materials for either biogas or ethanol production; however, improvement of biogas productivity or ethanol yield is necessary before an economical process can be achieved. r

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