Selection of thermotolerant Saccharomyces cerevisiae for high temperature ethanol production from molasses and increasing ethanol production by strain improvement.

Abstract

A thermotolerant ethanol fermenting yeast strain is a key requirement for effective ethanol production at high temperature. This work aimed to select a thermotolerant yeast producing a high ethanol concentration from molasses and increasing its ethanol production by mutagenesis. Saccharomyces cerevisiae DMKU 3-S087 was selected from 168 ethanol producing strains because it produced the highest ethanol concentration from molasses at 40°C. Optimization of molasses broth composition was performed by the response surface method using Box-Behnken design. In molasses broth containing optimal total fermentable sugars (TFS) of 200g/L and optimal (NH4)2SO4 of 1g/L, with an initial pH of 5.5 by shaking flask cultivation at 40°C ethanol, productivity and yield were 58.4 ± 0.24g/L, 1.39g/L/h and 0.29g/g, respectively. Batch fermentation in a 5L stirred-tank fermenter with 3L optimized molasses broth adjusted to an initial pH of 5.5 and fermentation controlled at 40°C and 300rpm agitation resulted in 72.4g/L ethanol, 1.21g/L/h productivity and 0.36g/g yield at 60h. Strain DMKU 3-S087 improvement was performed by mutagenesis using ultraviolet radiation and ethyl methane sulfonate (EMS). Six EMS mutants produced higher ethanol (65.2 ± 0.48-73.0 ± 0.54g/L) in molasses broth containing 200g/L TFS and 1g/L (NH4)2SO4 by shake flask fermentation at 37°C than the wild type (59.8 ± 0.25g/L). Among these mutants, only mutant S087E100-265 produced higher ethanol (62.5 ± 0.26g/L) than the wild type (59.5 ± 0.02g/L) at 40°C. In addition, mutant S087E100-265 showed better tolerance to high sugar concentration, furfural, hydroxymethylfurfural and acetic acid than the wild type.