Stress tolerance of Saccharomyces cerevisiae strains employed in the fuel ethanol production in Brazil.

Author(s)

Bianca Eli Della Bianca

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Escola Politécnica (EP/BC)
• Defense date: 2013-08-16

Examining board members

Andreas Karoly Gombert; Adriano Rodrigues Azzoni; Luiz Carlos Basso; Boris Juan Carlos Ugarte Stambuk; Aldo Tonso

• Advisor: Andreas Karoly Gombert

Abstract

As the need for biofuels rise, given their sustainable nature and the high prices of oil, so does the importance of the Brazilian fuel ethanol production in a global context. In Brazil, fuel ethanol is produced via fermentation of sugarcane feedstocks using robust strains of Saccharomyces cerevisiae. Understanding the physiology of these yeast strains has become the next necessary step to increase ethanol yields, since other major industrial processes in the ethanol production chain have already been significantly optimized. The aim of this thesis was to systematically evaluate the physiological responses of Brazilian fuel ethanol strains PE-2, CAT-1, BG-1, and JP-1, towards stress conditions associated to the process in which they are employed. Laboratory strains S288c and CEN.PK113-7D and bakers strain Fleischmann were also studied and considered as reference strains. Spot dilution assays in plates with a range of stressors in varying concentrations showed that industrial strains perform better under ethanol and acetic acid stresses and on industrial media (sugarcane juice and molasses). A distinction between fuel ethanol and bakers strains was observed only during growth under heat and low pH stresses, conditions that may be considered major factors of selective pressure in the fuel ethanol production environment, hindering the replication of the starter bakers strain. Heat stress was further studied in synthetic medium shake-flask cultivations at 37 °C, in which only laboratory strains exhibited a significant decrease on biomass and ethanol yields in relation to 30 °C. Carbon balance analysis showed that these strains channel more carbon to metabolites other than ethanol (like glycerol and organic acids), probably due to a higher triggering of stress response mechanisms under heat stress. Response of strains PE-2 and CEN.PK113-7D towards acidrelated stress conditions was analyzed in anaerobiosis in chemostats at pH 3.0 (on synthetic and rich media), in chemostats on synthetic medium added with 105 mM acetic acid and in dynamic continuous cultivations on synthetic medium with time-varying pH. In all these conditions both strains displayed similar physiology, with the exception of PE-2s particular acetate metabolism. In batch cultivations in rich medium at pH 2.7, however, remarkable differences could be noticedPE-2 strain exhibited a 33 % higher growth rate and an 86 % higher biomass yield. No differences between the strains were observed in batch cultivations in rich medium at pH 5.0 or in batch cultivations in synthetic medium at pH 5.0 or 2.8. Response to acid stress was also assessed in a non-proliferative environment, through measurements of cell viability after a 4-h H2SO4 treatment at pH 1.5. Strain PE-2 exhibited the highest viability (64.7 %), followed by strains Fleischmann (50.4 %) and CEN.PK113-7D (34.9 %). Analyzed together, the data presented in this thesis support the hypothesis that strain PE-2 was selected by surviving at low pH conditions found in the industrial cell recycle step, and by replicating fast in this stressful environment, most likely using dead bakers strain cells as a substrate. These features allow strain PE-2 to thrive in and dominate the fermentors in the fuel ethanol production process. (AU)

• FAPESP's process: 10/07187-0 - Tolerance of Saccharomyces cerevisiae towards stressors of the bioethanol industry
• Grantee: Bianca Eli Della Bianca
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)