PROBIOETHANOL - Proteomics and engineering of yeast ethanol tolerance for high-productivity bio-ethanol fermentation processes

Principal Investigator of the IST team: Isabel Sá-Correia

Contract: PTDC/BIO/66151/2006

Start date: 01/07/2007

Duration: 36 meses


Due to the continued reduction in oil supplies and the increasing costs of fossil fuels, there is an increasing interest in white biotechnology, which is aimed at producing chemicals or fuel replacements using durable renewable resources. Of the many different fossil fuel alternatives ethanol has become increasingly explored, since it can be used in combustion engines to levels of up to 20% without any modification to the engine. Furthermore it is considered as CO2 neutral, thereby having no net effect on greenhouse gas emissions. In order to produce the so called bio-ethanol, a cheap source of carbon and energy and other nutrients are required and a high productivity process is essential in order to minimize production costs. All bioprocesses require the presence of high cell densities in order to achieve high productivities. Furthermore continuous processes, particularly those with cell retention or cell recycle provide the highest productivities. One way to achieve this is to immobilize the yeast, or incorporate a cell recycle system, or use flocculent yeast cells, such that the majority of cells are retained during continuous operation. However, ethanol is toxic to the yeast above a critical concentration. The use of highly concentrated substrates will increase the ethanol productivity and minimize production costs as a significant decrease in the water handling and equipment capacity is achieved. Moreover, ethanol concentration in the fermentation broth will increase, decreasing distillation costs. Again, ethanol toxicity towards the microbial population hinders the improvement of ethanol productivity in the fermentation process. The aim of this project is to develop ethanol resistant yeast strains that are robust enough to be used in the ethanol fermentation from highly concentrated industrial based sugar substrates. The feasibility of using highcell –density continuous bioreactors with flocculent or non-flocculent immobilized yeast cells as a cost-effective ethanol production system will be evaluated. The project is designed so that we first determine the metabolic, physiologic, and genetic fundamentals underlying stress tolerance of ethanologenic yeast strains. This fundamental stress tolerance knowledge will then be used to engineer improved strains and/or design process conditions that foster stress tolerance and functionality of yeast cells for production of ethanol from highly concentrated industrial based sugars substrates, despite exposure to harsh environments. Success with engineered more stress-tolerant industrial yeasts will be combined with process optimization to yield a lower cost conversion of industrial sugars to ethanol. To achieve the aim of the project we will follow an integrative approach for strain and process development. Screening for phenotype differences in ethanol tolerance of a S. cerevisiae disruption mutant library will be the first step in the search for genes and gene systems fostering ethanol resistance. Using a proteome -wide analysis we will get insight in the molecular mechanisms responsible for ethanol resistance and will select mutant yeast strains to be characterize in respect to growth and alcohol production kinetics in highly concentrated substrates. Integrating the physiological characterization with the ethanol resistance phenotype we hope to be able to derive strategies to construct new yeast strains with improved ethanol resistance and fermentation ability. The ethanol resistance phenotype is highly dependent in environmental conditions. The whole project is designed so that yeast resistant strains will function well in simple and inexpensive growth media such as industrial based substrates. The constructed yeast strains and the designed fermentation medium will then be tested in both batch and continuous high-cell -density fermentation systems and its feasibility for bio -ethanol production evaluated.