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Saccharomyces cerevisiae strain selection for second generation ethanol production by fed-batch fermentation process with cell reuse


The aim of this Project is the selection of Saccharomyces cerevisiae strains with an adequate tolerance profile that allows 2G ethanol production from a lignocellulosic substrate composed by bagasse hydrolysate and cane molasses, using the fed-batch fermentation process with cell reuse. This process is widely used in Brazilian distilleries for 1G ethanol production, and the use of the same procedure and facilities could be an economic and reasonable alternative. The Brazilian fed-batch process allows very short fermentation time with high ethanol yield. Nevertheless recent work from our laboratory demonstrated that the most tolerant industrial strains available (namely CAT-1, PE-2 and SA-1) were not tolerant enough to perform fed-batch fermentations with cell recycling using the formulated lignocellulosic substrate. The great challenge for the implantation of this industrial process is that no Saccharomyces strains with the required multi-tolerance profile are available. In a first step ca. 500 indigenous Saccharomyces cerevisiae strains, isolated from Brazilian distilleries, will be screened for their tolerance towards the inhibitory action of the formulated substrate. The selected strains will be subjected to sporulation and the haploid pools will be used in mass mating. Simultaneously, isolated haploids will be screened for higher tolerance and used in directed crossings, searching for hybrids with even more robustness compared to parental. In a final step the selected tolerant strains will be engineered to construct strains able to convert xylose into ethanol using the CRISPR/Cas9 system. Finally, these multi-tolerant strains will also be valuable chassis for the insertion of metabolic attributes aiming the production of higher added value products other than ethanol. (AU)

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