The role of inositol in yeast adaptation

PhD Thesis


Ishmayana, Safri. 2019. The role of inositol in yeast adaptation. PhD Thesis Doctor of Philosophy. University of Southern Queensland. https://doi.org/10.26192/d1qg-sd55
Title

The role of inositol in yeast adaptation

TypePhD Thesis
Authors
AuthorIshmayana, Safri
SupervisorLynch, Mark
Learmonth, Robert
Kennedy, Ursula
Institution of OriginUniversity of Southern Queensland
Qualification NameDoctor of Philosophy
Number of Pages261
Year2019
Digital Object Identifier (DOI)https://doi.org/10.26192/d1qg-sd55
Abstract

Inositol is required for better yeast growth, stress tolerance and fermentation performance. This study focused on investigation of the role of inositol as a stress tolerance inducer, rather than as an essential growth factor. Three Saccharomyces cerevisiae strains with different properties were used in the present study, namely A12, A15 and K7. These strains were selected based on their different stress tolerance and ethanol productivity.

The first step of the present study was to determine the best conditions whereby the effects of supplementation of inositol can be differentiated from the control, where no inositol was added to the fermentation media. Since fluidity of the yeast cellular membranes was examined using fluorescence spectroscopy as one of the parameters in the present study, yeast nitrogen base (YNB) was used as basal media. YNB is a chemically-defined fermentation medium that does not interfere with fluorescence spectroscopy measurements in the UV to blue range. It is a useful medium for in situ monitoring, during fermentation, of cell physiology by fluorescence methods, however compared to rich media it is considered to have poor nutritional availability, which could affect the yeast ability to convert sugar to ethanol. Glucose concentrations ranging from 5 to 15% (w/v) were applied to investigate the highest concentration of glucose able to be efficiently converted to ethanol by each yeast strain. Growth and fermentation performance of the yeast strains were different. The fermentation performance could be ranked (highest to lowest) as strains A15, A12 and K7, while the growth performance could be ranked K7, A12, and A15. In general, fermentation with 15% initial sugar in the minimal medium led to lower sugar conversion to ethanol. The medium containing 10% glucose was considered the best to optimally differentiate fermentation performance of yeast strains.

The main objective of the present study was to investigate the effect of inositol supplementation on growth, fermentation performance and cellular membrane fluidity. Yeast cells were grown in a chemically-defined fermentation medium based on YNB but with no inositol and with 10% (w/v) glucose, and with varying levels of inositol supplementation (0.05, 0.10, 0.20, 0.40 and 0.80 g/L). Cell density, cell viability, glucose consumption and ethanol production were monitored for 96 hours. Cellular membrane fluidity was monitored at 24 hours fermentation, representing the respiro-fermentative growth phase, by measuring generalized polarization (GP) of laurdan. The effect of ethanol on membrane fluidity was also monitored by measuring GP after exposing cells to 18% (v/v) ethanol. When analysing the effect on fermentation kinetics it was found that inositol supplementation did not have the same effect on all strains, with A15 affected least. Although inositol supplementation did not seem to improve fermentation performance of yeast strain A15, it did improve cell growth leading to higher cell densities. Yeast strains A12 and K7 also evidenced higher cell densities with inositol supplementation, confirming the reported necessity of inositol for yeast growth. Unlike the preliminary experiment of the present study which used standard YNB medium containing 0.002 g/L inositol, the main experiments included analysis of multiple concentrations of inositol to better define the minimal requirement. While inositol-supplemented cells had higher growth rates and cell densities, they had significantly lower viability, thus the viable cell counts were similar with and without supplementation. Fluidity of the yeast cell cellular membranes responded differently to inositol supplementation. It was expected that inositol supplementation can increase membrane fluidity of all yeast strains. However, it was found that for yeast strains A12 and K7, inositol seemed to decrease cellular membranes fluidity, while for yeast strain A15 inositol supplementation led to increased membrane fluidity. When exposed to high ethanol concentrations, yeast strain A12 with inositol supplementation showed a significantly greater increase in membrane fluidity compared to cells grown without inositol.

Further analysis of the effect of inositol on fermentation performance of the three yeast strains showed that final ethanol concentration produced by yeast strain A15 was not significantly different when the fermentation media were supplemented with inositol, while yeast strains A12 and K7 produced significantly higher ethanol with inositol supplementation. Examination of the effect of inositol on yeast stress tolerance indicated that the three strains tested had better tolerance against ethanol, hyperosmotic and acetic acid stress when the fermentation media were supplemented with inositol. This led us to conclude that inositol acts as a general stress protector. One possible mechanism for the increased stress tolerance against hyperosmotic stress could be stimulation of synthesis of the osmoprotectant glycerol, as indicated by the significantly higher extracellular glycerol produced by inositol-supplemented cells. Inositol was also found to affect the fatty acids composition of the total cell lipid, where each strain showed a different response. No change was seen in the unsaturated fatty acid proportion for yeast strain A12, while yeast strain K7 showed a marked increase in C18:1, and yeast strain A15 had lower C16:1, but significantly higher C18:1.

In summary, the present study found that, compared to the industrial situation in which rich media are used, osmotic stress is evidenced at a lower sugar concentration (15% w/v, compared to 27%) when minimal media are used for fermentation. Therefore, to be able to distinguish the effect of inositol supplementation, we used a lower sugar concentration (10% w/v). At this concentration the sugar may not be exhausted, but the osmotic stress is not too severe. Inositol addition experiments indicated that even though inositol supplementation did not affect fermentation performance, it did increase cell growth and affect cellular membrane fluidity. Specifically, yeast strains A12 and K7 showed increased fluidity while A15 showed decreased membrane fluidity when grown in inositol-supplemented media. Further investigation on inositol supplementation indicated that inositol acts as general stress protector. Inositol supplementation also affects cellular fatty acid composition, where each strain showed a different response. Therefore, we conclude that inositol effects are strain-specific.

Keywordsyeast, fermentation, ethanol, cell tolerance, inositol, stress factors
ANZSRC Field of Research 2020310199. Biochemistry and cell biology not elsewhere classified
310603. Fermentation
Byline AffiliationsSchool of Sciences
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