Designing simultaneous saccharification and co-fermentation of lignocellulose for improved xylose conversion

Abstract: Fuel ethanol from lignocellulose is one sustainable alternative to the fossil fuels of today. All sugars in the material must be utilized in order to achieve high overall ethanol yields. Baker’s yeast, Saccharomyces cerevisiae, has been engineered to ferment the pentose sugar xylose from lignocellulose to ethanol. However, ethanol production from xylose is slow and often incomplete. In this work simultaneous saccharification and co-fermentation (SSCF) of xylose and glucose has been investigated with the purpose of improving xylose conversion and ethanol yields in non-detoxified lignocellulosic hydrolyzates. There are two main approaches to improve xylose conversion, which both have been investigated in this work. One way is to enhance the performance of the yeast by designing the process. The other way is to improve the yeast itself by genetic and/or evolutionary engineering. It was found that through careful design of different feeding strategies the xylose fermentation in SSCF could be significantly increased. Fed-batch, prefermentation, controlled enzyme feeding as well as combined enzyme and substrate feeding, had all positive effects on the xylose conversion. Depending on feed strategy and process conditions, this also resulted in a significant increase of the ethanol yield. Moreover, by designing the SSCF process, it was possible to increase the final solids content in the SSCF and still obtain a relatively high ethanol yield on total sugars, which is crucial for the process economy in commercial scale. The effect of improved xylose transport capacity in the yeast was investigated by expression of the glucose/xylose facilitator Gxf1 from Candida intermedia, in strains of S. cerevisiae which were assessed in SSCF. The improved transport proved to increase xylose uptake, but had only a minor effect on the ethanol yield. The enzyme xylose reductase (XR) was implicated to control xylose fermentation in SSCF of pretreated lignocellulose. When a mutated XR (mXR), with higher activity and altered co-factor preference, was integrated in S. cerevisiae the xylose uptake was significantly improved, resulting in a higher ethanol yield in comparison to when the native Pichia stipitis XR was used. Gxf1 had, however, only little influence when the combined effect of Gxf1 and mXR was studied in SSCF, which indicated that the initial xylose catabolism was still rate limiting.