Study of Resistance to Abyotic Factors through Oxidative Stress Biomarkers in Kluyveromyces Yeast
Authors: OCHOA BELEM VARGAS , Mejía‐Barajas Jorge A. , Arellano‐Plaza Melchor , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo 
Published: November 2023

There is an increasing interest in the production of bioethanol and the use of it as fuel due to the global energy crisis and the demand of environmentally friendly fuels. Bioethanol can be produced from lignocellulose as raw material because this material presents some advantages such as abundance and possessing large amounts of potentially fermentable sugars. However, during its production by the use of yeast, these cells face a variety of changes in the environment, such accumulation of ethanol and toxic compounds, mainly acetic acid and furfural, generating abiotic stress. This stress has been shown to favor the production of reactive oxygen species (ROS) and when their production prevails over the cellular defense system, the condition known as oxidative stress is generated, which also can result in damage to nucleic acids, proteins and lipids, mainly the unsaturated hydrophobic tails that form the lipids of the membranes. An important factor in cell acclimatization to environmental stress is the ability of cells to alter the degree of unsaturation in their membranes. Several authors have suggested a relationship between the fatty acid composition of phospholipid membranes and tolerance to some types of stress. Yeasts can incorporate a wide variety of exogenous fatty acids from the growth medium and these at the same time are rapidly incorporated into the lipids of the membranes. Therefore, it was of interest to study the relationship between resistance to abiotic factors and degree of unsaturation of the fatty acids of the yeast membranes of Kluyveromyces, through oxidative stress biomarkers. In the present work the resistance of the yeasts K. marxianus OFF1 and SLP1 against the acetic acid, ethanol and furfural inhibitors was determined by the method of MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide). Each inhibitor was added after growing yeast up to the stationary phase. K. marxianus OFF1 resists a greater concentration of acetic acid compared to K. marxianus SLP1. In the case of ethanol both yeast resist 10% reducing its viability to 50% at 6 and 7 hours, respectively. Furfural 30 mM reduced the viability of K. marxianus SLP1 by 50% in 2 hours, while K. marxianus OFF1 viability was reduced at the same percentage at 8 hours and 20 mM. Viability in both yeasts was improved and restored to around 100% by the addition of 1 mM of oleic, linoleic, linolenic and arachidic fatty acids in separate way at the IC50 of ethanol and furfural for each yeast. The rate of lipoperoxidation was determined by the reaction with thiobarbituric acid. The lipoperoxidation of yeasts treated with each inhibitor was reduced when it was added each of the fatty acids mentioned above. Finally, the level of carbonylation was determined by the reaction of 2,4‐dinitrophenylhydrazine and there was no changes by the addition of each fatty acid to yeast treated with each inhibitor, respect to not treat. In conclusion, the addition of fatty acids to the yeast favored its resistance to the inhibitors furfural and ethanol.

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Special thanks for the grant of CIC‐UMSNH:2.16 to ASM; BVO is CONACYT fellowship.