Selection and improvement of Saccharomyces cerevisiae by direct and mass mating for integrated first and second generation (1G + 2G) ethanol production

Abstract

The integrated 1G + 2G ethanol production process seems to be a feasible strategy considering the Brazilian scenario, in which sugarcane molasses and bagasse are available substrates in biorefineries. However, such process imposes harsh conditions to yeasts, which must be able to simultaneously deal with several stresses from molasses and hydrolysate. In this work, improved hybrid strains of Saccharomyces cerevisiae with higher resistance for an integrated 1G + 2G ethanol production were obtained by hybridization technique. Initially, 663 wild S. cerevisiae strains were screened in microculture growth assays. Seven strains were pre-selected out of 40 based on their stress multi-tolerance traits. Subsequently, these pre-selected strains underwent evaluation in cell-recycling fed-batch fermentation conditions utilizing a medium containing molasses-bagasse hydrolysate. The two most successful strains from this evaluation were subjected to sporulation, yielding haploid cells. These haploid cells were then subjected to crossbreeding, employing direct and mass mating techniques. From the initial pool of 7 pre-selected strains, 3 hybrid strains were selected due to their enhanced resistance under the demanding conditions of cell recycling fed-batch fermentation, particularly when utilizing a higher osmotic molasses-hydrolysate medium. These hybrid strains consistently exhibited high cell viability rates and effectively accumulated elevated levels of trehalose and glycogen over the course of five fermentation cycles. Remarkably, they maintained ethanol productivity at levels comparable to their respective parental strains. The genotyping molecular identification attested the difference between the hybrids and the parental strains. The 3 hybrids selected seem to present valuable backgrounds for metabolic engineering for lignocellulosic-derived xylose fermentation.