| Abstract | Ethanol tolerance is one of the most important properties of yeasts used for bioethanol production, and has been correlated with plasma membrane fluidity. This study investigates yeast membrane fluidity and ethanol tolerance, particularly in relation to proline and inositol supplementation. Three Saccharomyces cerevisiae strains (A12, PDM and K7) were selected, based on reported stress tolerance and ethanol productivity; an ethanol tolerant baker’s yeast (A12), a wine yeast (PDM) and a sake yeast (K7), the latter produce up to 17 and 17.5 %(v/v) ethanol, respectively.
To determine the feasibility of these strains and supplementation for bioethanol production, a model system was devised using Yeast Nitrogen Base (YNB) with 18% (w/v) sucrose. YNB was chosen for its defined and consistent composition (limiting variation) and for its lower fluorescent background (enabling membrane fluidity assessment in situ). However growth of all strains was inconsistent and ferments stuck at high sugar levels. This was likely due to insufficient nitrogen or other essential nutrients, and could be ameliorated by a complex but undefined medium but with high and inexact levels of proline and inositol. In order to allow unequivocal discrimination of supplement effects, experiments were continued with media similar to previous laboratory studies; YNB with 2% (w/v) glucose.
When cultured in YNB with 2% (w/v) glucose, the three strains had similar growth rates and performance, although K7 maintained significantly higher viability. Comparison of generalized polarization (GP) of laurdan-labelled cells indicated that PDM had the highest membrane fluidity, followed in order by K7 and A12. Conversely A12 had the highest ethanol tolerance, followed in order by K7 and PDM, so unlike some published reports, higher ethanol tolerance related to lower membrane fluidity. Furthermore in comparison to 6 h cultures, 24 h cultures of all strains had lower membrane fluidity and higher ethanol tolerance.
Two approaches were used to assess ethanol tolerance. The total plate count (TPC) is widely used to assess ethanol tolerance, while methylene violet staining has been proposed as a rapid alternative. Correlation analysis showed only weak correlations between viability assessment by methylene violet staining and viability by TPC, membrane fluidity by GP or culture age. In contrast there were strong correlations between membrane fluidity by GP, viability by TPC and culture age.
Despite showing promise in previously published studies as a stress tolerance enhancer, proline supplementation did not lead to any consistent significant change in membrane fluidity or ethanol tolerance. The only significant effect was the higher GP of the PDM strain with 0.5 g/L proline. However, no significant differences between levels of supplementation were detected in viability reduction in ethanol-stressed cultures (either by TPC or methylene violet staining). Therefore further study is needed to confirm this result. The present study failed to confirm reports that inositol supplementation increases ethanol tolerance. No significant changes of either GP or viability reduction upon ethanol stress were found when the medium was supplemented with various levels of inositol. Further investigation, including more variations in concentration, is needed to elucidate this possibility.
In summary, of the three S. cerevisiae strains tested, A12 seems to be the best for bioethanol production, followed by K7 and then PDM. Some relationships were found between culture age, ethanol stress tolerance and membrane fluidity, although supplementation of cultures with proline or inositol did not seem to enhance culture performance or ethanol tolerance. |
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