The effects of thermal and ethanolic stress in industrial strains of Saccharomyces cerevisiae

Saccharomyces cerevisiae is exceptional microorganisms used in biotechnological processes, mainly in the ethanol production chain. Studies of the cellular responses of industrial yeasts under ethanolic and thermal stress in an association are still incipient. This study aimed to evaluate the action of thermal and ethanolic stress in industrial strains of Saccharomyces cerevisiae under different temperatures and concentrations of ethanol, to understand whether these factors influence ethanol production. For cytotoxicity and genotoxicity tests, yeasts were grown in 2% YPD medium incubated for 10 hours at 250 rpm. After growth, the Research, Society and Development, v. 9, n. 10, e6819109091, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.9091 2 samples were grown in sugarcane juice in concentrations of 5, 10 and 15% ethanol and incubated at 30 and 40 oC. In Petri dishes containing the solid medium YPD 2% the yeasts were dripped and incubated for 72 hours the cytotoxic action was analyzed by cell growth and genotoxicity through the comet assay and ethanol production by gas chromatography. Cell growth occurred in all conditions, however, at 30 oC there was inhibition in 10% (v v) of ethanol being potentiated in 15% (v v), at 40 oC. The genotoxicity analysis showed an induction of DNA damage in yeasts, however, the FLE yeast was the one with the highest DNA damage index. The yeast Pedra-2 was more tolerant and produced more ethanol, showing to be a tolerant strain concerning the analyzed fermentative interferents.

reduction in the viability rate, which in turn leads to slow fermentations and the accumulation of toxic by-products that induce biological responses and alter productivity throughout the industrial process (Vargas-Trinidad et al., 2020). Thus, the fermentation of ethanol at high temperatures is desirable, since it has advantages such as the reduction of contamination and reduction of cooling costs (Abdel-Banat et al., 2010), although they can cause physiological changes in yeasts.
Ethanol varies approximately between 8% and 10% in the fermentation medium (Cray et al., 2015) is an alcohol formed by two carbons which, due to its short alkene chain and the hydroxyl group, is soluble in water and lipids, and thus it can cross the plasma membrane producing an increase in fluidity and alteration of the membrane permeability, in addition to modifying the biosynthesis of macromolecules and causing greater production of heat shock proteins and increased mutations (Morard et al., 2019;Hu et al., 2009). In addition, the accumulation of extracellular ethanol causes a restriction in the excretion of intracellular ethanol that causes a suppressed production effect (Cao et al., 2020).
The multiple industrial stressors to which yeasts are exposed can cause damage to deoxyribonucleic acid-DNA, cease the cell cycle, cause abnormal enzyme production, among other molecular changes (Brown & Kobor, 2019). In addition to these external agents, cell metabolism itself generates thousands of daily lesions, which alter the double helix of DNA and cause instability of the genome that can compromise replication and transcription and consequently result in mutations during the S phase, or chromosomal aberrations when there are breaks in the DNA (De Andrade Lima, 2015). Cytotoxic analyzes are essential to prevent harmful interference to yeasts in the industrial context, in addition to identifying the harmful capacity of compounds harmful to these living beings. Genotoxicity must also be taken into account since it covers processes that alter the chemical and physical structure of DNA (Santos, 2019).
The ethanol tolerance phenotype is complex and also influenced by external factors, mainly as temperature, in many studies, there is an inherent interest in the particularity of responses involving similar genes that thermal and ethanolic stress induce and some even demonstrate that tolerance to a factor is dependent on another (Fujita et al., 2006;Caspeta et al., 2019). In this sense, research is aimed at understanding the mechanisms of tolerance of these microorganisms in the face of thermal and ethanolic stress. But, most of these studies look at factors separately and not their associations (Riles & Fay, 2019). Therefore, this theme is still not completely elucidated because it is a characteristic of multiple loci and their related genes are randomly distributed in the genome (Giudici et al., 2005). Development, v. 9, n. 10, e6819109091, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.9091 5 In this bias, studies are becoming important to understand the respective effects of the action of ethanol associated with high temperature and how they trigger lesions in yeast cells and genetic material. Thus, this study aimed to evaluate the action of thermal and ethanolic stress in industrial strains of Saccharomyces cerevisiae at different temperatures and concentrations of ethanol, aiming to understand how these stress factors can cause cellular changes and consequently influence the production of ethanol, an important biotechnological product.

Research location
The study was developed at the Biotechnology, Biochemistry and Biotransformation laboratory of the Center for Studies in Natural Resources-CERNA of the State University of Mato Grosso do Sul-UEMS / Dourados-MS.

Microrganisms Used
The microorganisms used for this study were Saccharomyces cerevisiae FT-858 obtained from the company Fermentec located in Piracicaba -SP, Pedra-2 (Pe-2) acquired in the company LNF Biotecnologia Aplicada, located in Bento Gonçalves -RS and the yeast Fleischmann® (FLE) acquired commercially.

Pre-inoculum
The pre-inoculum consisted of 2% YPD medium containing 1.0% (w v -1 ) of yeast extract; 1.0% (w v -1 ) peptone; 2.0% (w v -1 ) glucose, sterilized in an autoclave at 120 ºC for 20 minutes, in which 0.10 grams of lyophilized yeasts were inoculated and incubated at 30 ºC for 10 hours at 250 rpm. After this period, the cells were collected by centrifugation, resuspended and washed with sterile saline.

Fermentative Condition
For the fermentation experiment, 50 mL of the sterile cane juice was added in 125 mL Erlenmeyer flasks. After 10 hours of growth, the biomass obtained was inoculated in the Research, Society and Development, v. 9, n. 10, e6819109091, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.9091 6 fermentation medium in the presence of the stressor ethyl alcohol in the concentrations of (5%, 10% and 15%) and the samples were incubated at 30 ºC and 40 ºC for 10 hours at 250 rpm . For the negative control, there was no addition of ethanol and for the positive control, in the genotoxicity test, 3% hydrogen peroxide was added.

Cytotoxicity Test
The stress-induced samples, as described in item 2.4, were added in a Petri dish containing the solid medium YPD 2%, 5 μl of the samples were dripped and incubated at 30 ºC for 72 hours. The results were analyzed concerning cell growth capacity in the face of different concentrations of ethanol and temperature.

Genotoxicity Test
The comet assay was conducted according to Mueller et al. (2019)

Quantification of Ethanol
The analysis of the ethanol concentration was determined with a gas chromatograph (CG 3900) with flame ionization detector (Varian), using a 30m long fused silica capillary column (ZB-5). The chromatographic condition used was an injection volume of 1 μL, split ratio 1:20 and oven temperature of 90 ºC. The samples were filtered through a 0.22 μm production, in addition to toxicity, high temperatures, pH and osmolarity are also recognized as tensions to be overcome during the process. Such an occurrence could be observed by the phenotypic profile presented by the yeast FLE, which proved to be more sensitive to the action of inhibitory agents. Figure 2   In the fermentative niche, stressors can limit the development of other microorganisms, as well as that of the yeast itself and in this context, this inhibition can be considered cytotoxic (Cray et al., 2013). Selected yeasts can tolerate an anoxic environment, with high temperatures and high alcohol content that is generated during industrial fermentations, better than wild yeasts and this fact can be considered a selective advantage. In this sense, high concentrations of ethanol in the fermentation medium can be potentially harmful (Goddard & Greig, 2015). Research, Society and Development, v. 9, n. 10, e6819109091, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.9091

Genotoxicity
From the genotoxicity analyzes through the comet assay it was possible to observe that there was the induction of lesions in the deoxyribonucleic acid -DNA of the yeasts analyzed in all ethanol concentrations and at both temperatures. Despite this, at 30 ºC the lowest injury rate was observed at 5% in the Pe-2 strain and the second-lowest for FT-858 at the same concentration, while the FLE yeast showed the greatest sample damage at a concentration of 15% at the same temperature. mentioned. At 40 ºC yeasts showed a damage profile similar to that described for the previous temperature, however, with an increase in lesions in the concentrations of 10 and 15% mainly for the lines Pe-2 and FT-858, however, the FLE was more susceptible in relation to the other strains, especially in the highest concentration of ethanol ( Figures 3A and 3B). Eventually, only the ethanol factor alone has already caused lesions sufficiently harmful to the cells, especially for the FLE strain, which showed greater sensitivity to this stress factor. However, the action of elevated temperature associated with a high concentration of ethanol was able to generate a higher damage index in the analyzed yeasts, which is probably related to a profile of distinct physiological responses that each yeast presents in relation to the separate damage and in synergism. It is worth mentioning that the toxic action of ethanol and the high temperature was also observed in the results presented of A B Development, v. 9, n. 10, e6819109091, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.9091 11 the cell growth capacity in relation to the studied yeasts. The tolerance profile given to Pe-2 may be related to the fact that this strain has a lower amount of DNA-induced damage that characterizes a lesser genotoxic action of ethanol compared to this strain.
The changes that occur in the course of industrial fermentations, most of the times unfavourable, require adequate cellular responses and among the several existing mechanisms of cellular protection that are activated in response to stressful conditions, we can mention the cross-protection against different stresses, in which the cells exposed to a light dose of a stressor respond in a resistant way to larger and generally lethal doses of others (Swiecilo, 2016).
In addition, stress response elements (STREs) control responsive genes in response to different stresses, including heat shock and high concentrations of ethanol through transcriptional factors like Msn2p and Msn4p that modulate protein kinase-dependent gene expression (PKA), however, are activated in different ways and induce the transcription of a large battery of genes containing STRE, which will mediate the overall stress response in yeasts (Estruch, 2000;Saini et al., 2018). Studies by Riles & Fay (2019), analyzed the genetic basis of ethanol tolerance in Saccharomyces cerevisiae at elevated temperatures and showed that polymorphisms of some genes can cause sensitivity or tolerance to ethanol at high temperatures. As is the case with two amino acid substitutions in the SEC24 gene region, which underlies the locus that gives the yeast thermotolerant characteristics.
In this study, we can observe that the induction of the DNA damage index was more significant when the stress factors were associated in relation to the strains analyzed. This fact may be related to the hostile conditions that frequently occur during fermentation processes, causing yeasts to present intermittent physiological mechanisms of responses to such adverse conditions. Therefore, our data corroborate with the literature, because it was observed that yeasts had a functional profile of different inductions of genetic lesions in relation to the associated stressors. Possibly, the yeast response mechanisms are not a singular event, but the interactivity of several genes involved forming a systematic and complex network in the intracellular environment leading to cytological and genetic changes.

Ethanol Production
In the evaluation of the concentration of ethanol, it was possible to observe differences in the production profile of this compound concerning the yeasts at the analyzed temperatures.
Higher production of this metabolite (9.0 v v -1 ) was observed at a temperature of 30 ºC for