Osmotic adjustments, correlated with changes in water availability, is one of the major causes of stress for cellular systems. Osmotic stress can be induced by high levels of extracellular solutes (such as glucose) and reduce cell turgor. Micro-organisms can perform 'osmoadaptation' mechanism producing compatible solutes (such as ions, amino acids or polyols), which protect proteins and nucleic acids, increasing the osmotic pressure of the cytoplasm. Polyols (for example erythritol and glycerol) are low-molecular weight solutes, synthesizes and/or accumulated, and then secreted, by yeasts and fungi at low aw ('water activity' which describes the (equilibrium) amount of water available for cells). Each micro-organism has a specific polyol-pool, which changes in function of experimental conditions. We are interested in examine this microbial process of defense and compatible solutes production. This work can be divided into four main sections:
I- Selection of the microrganism:
First of all we analyzed several 'osmotolerant' yeast species for their high resistance to glucose concentrations, searching for the phenotypic trait of 'osmoadaptation' (the specie which produces high biomass levels in less time). We confirmed selection by design of experiment method and we observed that the same fungus gave us the best erythritol yields during transformation of glucose trough aerobic fermentation. We characterized physiologically this dimorphic basidiomycetous sugar-tolerant yeast and we analyzed different conditions for polyol production. Media composition resulted the most important factor influenced polyols (and in particular erythritol and glycerol) concentration.
II- Optimization of culture medium composition:
We selected the nutrients to add in the basal medium as key parameters to affect polyol production; then we observed responses studying the relationships with the nature and concentration of used sources. Experimental design (DOE) composition method made it possible to determine the optimum medium composition for biomass and polyol productions. We chose different independent variables (or factors), each at different levels (specific area of interest), and analyzed responses after elaboration of specific models. Each experiment was statistically planned and carried out on flasks. The cultivation parameters (such as pH, agitation, temperature) were kept constant, using a standardized inoculum (10% v/v, grown for two days).
III- Microbial process development:
Statistical method of response surface methodology has been applied to analyze the experimental data with the aims of enhancing fermentative production of erythritol and controlling the production processes. Interested in a possible industrial application, we established and performed a two-stages process of feed-batch fermentation, using 2L and 20L bioreactors.
IV- Improvement of yeast strain:
We use statistical experiments also for screening mutants obtained after random UV-mutagenesis of wild-type strain and selected for its best 'osmotolerant' phenotype (resistance in presence of glucose up to 60%). To improve its performance, we are also trying to transform this fungus by electroporation.
These results should be a useful tool for further potential investigations.
