Inducing yeasts dominance in the presence of multiple environmental stress to cream off new robust candidates for lignocellulosic bioethanol production

Increasing attention has been recently devoted to the production of bioethanol from lignocellulosic biomass. However, lignocellulose is expensive to process because of the need for costly pre-treatments and large dosages of commercial enzymes. Moreover, lignocellulose pre-treatment results in the formation of inhibitors affecting the following fermentation phase. Although ethanol production from pretreated lignocellulose has been widely described (Hamelinck et al., 2005), only limited efforts have been spent on selecting yeasts able to both tolerate inhibitors and ferment sugars (Lindén et al., 1991; Favaro et al., 2012). This study aimed at the isolation, characterization and selection of robust yeasts suitable for the lignocellulose-to-bioethanol route. While the majority of related researches has focused on isolating yeasts from first generation bioethanol and/or wine industrial plants, this work specifically targets to yeasts capable of exhibiting their dominance once challenged by multiple environmental stress all at once. To this purpose, grape marcs were selected as extreme environment because of its limited nutrients, exposure to solar radiation, temperature fluctuations and ethanol (Favaro et al., 2013a). Grape marcs, collected, immediately after crushing, from a winery, were left at 30 and 40 °C for seven days, afterwards samples have been incubated at both temperatures in fermenting bottles containing 100 mL YNB (Yeast Nitrogen Base) broth supplemented with 100 g/L glucose, 50 g/L xylose and a cocktail of inhibitors (aliphatic acids, furans and aldehydes). Microbial biomass fluctuations together with ethanol and by-products concentrations have been monitored by means of microbiological techniques and HPLC (High Performance Liquid Chromatography) analysis. As soon as ethanol productions and biomass concentrations reached reasonable levels, samples of each fermenting bottles were aseptically transferred in fresh YNB formulated with the same sugars composition but with higher levels of inhibitors. After three month experimental activities, the evolved yeast populations were able to grow quite fast in the presence of elevated inhibitors concentrations exhibiting promising ethanol yields. Moreover, microbiological quantitative and qualitative analyses, performed at the beginning of the experiment and periodically throughout all the transfers, gave a fascinating picture of population dynamics as well as of yeast species dominance in such hostile environment. At 30 °C, the majority of the isolates capable of withstanding osmotic, ethanol and inhibitors stress belonged to Saccharomyces cerevisiae while, at 40 °C, the dominant species revealed to be Issatchenkia orientalis. On the other hand, when higher inhibitor levels were added, lower consistency of I. orientalis was detected in favour of S. cerevisiae. The isolated yeasts were then screened for their fermentative abilities in minimal medium as well as for their inhibitors tolerance as described by Favaro et al. (2013b). Interestingly, many S. cerevisiae strains exhibited very high ethanol yields also at 40 °C and displayed strong abilities to tolerate weak acids, furans and aldehydes. The preliminary results of this study indicate that the obtained microbial collection is a promising platform towards the development of robust and efficiently fermenting microbes suitable for the industrial processing of lignocellulosic biomass into ethanol.