Towards a Kluyveromyces marxianus strain suitable for non-aseptic fuel ethanol production at high temperature

Abstract

Brazilian ethanol production is one the cheapest and fastest industrial bioprocesses in the world. However, improvements to the fermentation process are necessary to maintain a profitable business. The yeast Saccharomyces cerevisiae presents the most suitable features to produce ethanol under industrial conditions: high ethanol tolerance (concentrations higher than 10%); ability to grow under complete anaerobiosis; high survival at low pH (< 2.5) (to enable cell recycling under acidic conditions). However, two important challenges are recognized during fermentation: temperature and contamination. The process is performed at 32 to 35 °C, requiring cooling of the fermentation vats with cold water. In addition, because contaminants are present (the process is not aseptic), an acid treatment (pH < 2.5 for 2-3 h) must be carried out during the cell recycling step, to decrease the bacterial load. One alternative would be high-temperature ethanol production, which would reduce contamination risks, as well as the costs and the time to carry out the cooling and distillation processes. A microorganism that can potentially fulfill this feature is the yeast Kluyveromyces marxianus. K. marxianus also presents a high specific growth rate, both fermentative and respiratory metabolism, the ability to metabolize a wide variety of sugars, and a highly developed and efficient genetic toolbox, which enables virtually any desired genetic modification to be implemented into this yeast species. To date, however, there is no single K. marxianus strain reported to produce ethanol under full anaerobiosis and to tolerate high concentrations of ethanol and low pH. In the present proposal, we will apply evolutionary and metabolic engineering to K. marxianus, in order to generate a yeast strain that can produce ethanol at 48°C, under the same conditions used in the current Brazilian ethanol industry. (AU)