Comparative study of flocculent and non-flocculent yeast strains for ethanol production

Abstract

The gradual depletion of crude oil and the biological environmental deterioration resulted from both the fuels over consumption and petroleum-derived transportation have gained attention again, making urgent to develop renewable and environmentally friendly alternatives. Ethanol is an important industrial chemical with emerging potential as a biofuel to replace vanishing fossil fuels, whose utilization could improve energy security and decrease urban air pollution. The entry into force of Law 26,093 of biofuels in Argentina from 2010 will mean an opportunity for the sugar sector to expand ethanol production in order to supply 5% of this alcohol to all the naphthas. Our work proposes a microbiological approach to use fermentative microorganisms with high tolerance to alcohol in order to increase the currently obtained ethanol concentration (11%) by the alcoholic fermentation of molasses and compare the results when using flocculent or nonflocculent yeast strains for fermentation. To take up this, isolation and identification of ethanol hyper-producing yeasts strains from sugar cane molasses was carried out. Samples of molasses were taken from different mills of Tucuman and used to inoculate YPS (w/sucrose), YPD (w/dextrose) and molasses media with antibiotics. YPS medium with 50g/L sucrose was used for the propagation of microorganisms by incubating in a thermostatically controlled bath at 30ºC with agitation. Fermentations of selected isolates were performed in duplicate in flasks with 200 ml of YPS medium with 250 g/L sucrose and incubated at 30ºC without aeration. Every 8 h, Total Reducing Sugars (TRS), Direct Reducing Sugars (DRS), biomass dry weight and ethanol concentration were determined. Three yeast isolates showing high ethanol production and named as A2, A10 and A11, which produced 11.74, 12.81 and 13.20% ethanol, respectively, were selected. A10 and A11 were flocculent yeast strains and A2 a non-flocculent yeast strain. These isolates were identified by molecular taxonomy tools according to the sequence analysis of their rDNA intergenic spacers, which allowed assigning identities of 99 and 100% to that of Saccharomyces cerevisiae. Fermentations were carried out with a massive inoculum, which allowed to reduce the time of fermentation, and in the case of the A2 strain an ethanol concentration of 11.95% could be reached after 10 h of incubation, a higher value than the one currently achieved in industry, which is 10%. It was determined that the strain A2 showed an homogeneous growth in liquid media, a feature that is compatible with the technology used in industry. For this reason, the A2 strain could be used in ethanol industrial production without the need for technology investments that would be indispensable for the recycling of flocculent strains. However, the A10 and A11 flocculent yeasts showed a very important potential for ethanol production, and these promising results justify further studies leading to an optimization in the production of bioethanol.