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Abstract Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

Abstract Background High acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates. It is also important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. We have previously identified snf4 E269* as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance. Results We have now performed polygenic analysis with the same WG transformant MS164 to identify novel causative alleles interacting with snf4 E269* to further enhance acetic acid tolerance, from a range of 0.8–1.2% acetic acid at pH 4.7, to previously unmatched levels for Saccharomyces cerevisiae. For that purpose, we crossed the WG transformant with strain 16D, a previously identified strain displaying very high acetic acid tolerance. Quantitative trait locus (QTL) mapping with pooled-segregant whole-genome sequence analysis identified four major and two minor QTLs. In addition to confirmation of snf4 E269* in QTL1, we identified six other genes linked to very high acetic acid tolerance, TRT2, MET4, IRA2 and RTG1 and a combination of MSH2 and HAL9, some of which have never been connected previously to acetic acid tolerance. Several of these genes appear to be wild-type alleles that complement defective alleles present in the other parent strain. Conclusions The presence of several novel causative genes highlights the distinct genetic basis and the strong genetic background dependency of very high acetic acid tolerance. Our results suggest that elimination of inferior mutant alleles might be equally important for reaching very high acetic acid tolerance as introduction of rare superior alleles. The superior alleles of MET4 and RTG1 might be useful for further improvement of acetic acid tolerance in specific industrial yeast strains.

Abstract Background The search for sustainable energy sources has become a worldwide issue, making the development of efficient biofuel production processes a priority. Immobilization of second-generation (2G) xylose-fermenting Saccharomyces cerevisiae strains is a promising approach to achieve economic viability of 2G bioethanol production from undetoxified hydrolysates through operation at high cell load and mitigation of inhibitor toxicity. In addition, the use of a fixed-bed reactor can contribute to establish an efficient process because of its distinct advantages, such as high conversion rate per weight of biocatalyst and reuse of biocatalyst. Results This work assessed the influence of alginate entrapment on the tolerance of recombinant S. cerevisiae to acetic acid. Encapsulated GSE16-T18SI.1 (T18) yeast showed an outstanding performance in repeated batch fermentations with cell recycling in YPX medium supplemented with 8 g/L acetic acid (pH 5.2), achieving 10 cycles without significant loss of productivity. In the fixed-bed bioreactor, a high xylose fermentation rate with ethanol yield and productivity values of 0.38 gethanol/gsugars and 5.7 g/L/h, respectively were achieved in fermentations using undetoxified sugarcane bagasse hemicellulose hydrolysate, with and without medium recirculation. Conclusions The performance of recombinant strains developed for 2G ethanol production can be boosted strongly by cell immobilization in alginate gels. Yeast encapsulation allows conducting fermentations in repeated batch mode in fixed-bed bioreactors with high xylose assimilation rate and high ethanol productivity using undetoxified hemicellulose hydrolysate.