Ability of The Saccharomyces Cerevisiae Y904 to Tolerate and Adapt to High Concentrations of Selenium

The rational use of by-products is essential for the development of a sustainable society. Worldwide, the alcoholic fermentation industry generates a large surplus of yeasts, on the scale of millions of tons. So there is a need for benecial applications to humanity of this surplus. Yeasts, in turn, have the ability to bioaccumulate minerals and enable their bioavailability after cell autolysis. Among these minerals, we highlight selenium (Se), which participates in the formation of antioxidant enzymes. The objectives of the work were to dene the minimum and maximum concentration of Se that yeasts (Saccharomyces cerevisiae – Y904) support and the concentrations that they tolerate once adapted. To this end, a test of tolerance to Se was carried out, using treatments with different concentrations of Se. The adaptive process started at the maximum concentration obtained in the tolerance test of 60 µg mL − 1 , with an increasing addition of 6 µg mL − 1 , reaching up to 246 µg mL − 1 of Se. The macromorphological characteristics and number of colony forming units were evaluated. It was identied that yeasts without adaptation grew on substrate containing up to 60 µg mL − 1 of Se and those adapted, up to 246 µg mL − 1 of Se. In addition to the reduction in yeast growth speed, from the concentration of 84 µg mL − 1 of Se in the medium, morphological changes in colony color were observed. It is concluded that non-adapted yeasts support up to 60 µg mL − 1 of Se and, after the adaptive process, they support 246 µg mL − 1 of Se in the medium.


Introduction
Brazil is currently the second largest producer of fuel ethanol in the world, generating approximately 33 billion liters per year (Nova cana 2020). In the ethanol production process, at the end of each fermentation cycle, there is a large surplus of yeasts of the Saccharomyces cerevisiae species, around 20 kg of yeasts per m 3 of ethanol produced, which generates about 660,000 tons (DESMONTS 1966). These single-celled microorganisms have the ability to transform sugars into ethanol, carbon dioxide, energy and other by-products. In the ethanol production process, yeasts are used only as agents of biotransformation of sugar into ethanol, and after this biochemical reaction, they can be reused for other purposes, such as enrichment in nutrients of interest (MUSSATTO 2010). In some situations, while the yeast cell recycling, after yeast treatment, part of them can be removed from the process and enriched with minerals, such as Se (SUHAJDA et al. 2000;BASSO et al. 2008). As they also have the ability to bioaccumulate many chemical elements, yeasts are used as sources of micro and macronutrients for human supplementation. Among these nutrients, Se can be highlighted, which is a micronutrient that participates in several antioxidant metabolic routes in human body (RIAZ et al. 2012).
According to TINGGI (2003), Se participates in the conversion of triiodothyronine hormone (T3) into thyroxine hormone (T4) and in the action against toxic and xenobiotic metals. It also participates in the prevention of chronic and non-communicable diseases, in the ubiquinone biosynthesis, which is an important biological process, and it is an essential nutrient for animals and humans that can be obtained as a source for yeast enrichment.
The yeast S. cerevisiae used in alcoholic fermentations has the ability to transform inorganic Se into organic compounds, which facilitates its bioavailability in the body and, depending on its growth conditions, it can accumulate remarkable amounts of Se in the form of selenomethionine and selenocysteine (PEDRERO et al. 2009). The organic forms of Se are part of the active site of important selenoproteins, such as glutathione peroxidase, which act to contribute to cell homeostasis by ghting free radicals.
For these reasons, the objectives of this study were to evaluate the tolerance of yeast S. cerevisiae to high Se concentrations; to carry out the evolutionary adaptation of yeasts to this mineral and to investigate the morphological variations of yeasts colonies / cells according to the evolutionary adaptation.

Material And Methods
Testing location: The tests were carried out at the Sugarcane and Bioenergy Technology Laboratory (LTSBio), Sugar and Alcohol Sector, Department of Agribusiness, Food and Nutrition, of ESALQ / USP.
As a criterion for analyzing tolerance, yeast growth was considered up to 48 hours of incubation, after inoculation in a Se-rich medium. Thus, yeasts growing under these conditions were considered tolerant and those that did not grow were considered susceptible to Se.
Adaptation study of Saccharomyces cerevisiae in a medium enriched with sodium selenite: From the results obtained in the tolerance study, the maximum dose of the nutrient in which the yeasts managed to grow was selected. Then the adaptation process of the yeast S. cerevisiae Y904 was started in a culture medium enriched with sodium selenite (Na 2 SeO 3 ) ACS QM ® , 99.0% purity.
The rst adaptive cycle was performed with YEPDA enriched with 60 µg mL − 1 of Se. Subsequently, gradual increases in Se concentrations of 6 µg mL − 1 per cycle were performed during 32 consecutive culture cycles. The adaptation process started with 60 µg mL − 1 (D1) and in cycle 32, the concentration of Se in the culture medium was 246 µg mL − 1 (D32).
Petri dishes contained 10 mL of substrate and received 100 µL of inoculum, with dilutions of 10 − 5 and 10 − 6 CFU mL − 1 . The incubation was carried out at 30 ± C ± 2 ˚C, for 24 to 48 hours, according to the colonies growth and to the methodology described by ASSUNÇÃO (2011). As the colonies grew, those that grew within the 48-hour period were considered adapted and those that did not grow were considered susceptible. The analyzed macromorphological characteristics of the colonies / cells were: color, size, odor and roughness.
The biomass of each treatment was stored in a 0.5 mL microtube containing the modi ed Karnovisky reagent, composed of 2.5% gluraldehyde, 2.5% formaldehyde 0.05M, sodium cacodylate buffer solution at pH 7.2, and CaCl 2 0.001 M. The samples preparation followed the protocol of KITAJIMA (1999), in which a drop of poly-L-lysine was added to the coverslip and this was placed to rest for 15 to 20 minutes, then a sample drop was added in suspension keeping at rest for more 30 minutes in the coverslip. The coverslips with separators between one sample and another were placed in the "cage" to dehydrate inside the "cage" in a beaker in increasing concentrations of acetone: 30%, 50%, 70% and 90% for 30 minutes at each concentration, and 100%, 3 times of 30 minutes each, to then be dried to the critical point, using CO 2 . Finally, the coverslips were xed in the stubs submitted to the metallization process, so that the samples could be observed and analyzed in the SEM with an increase of 10,000 times.

Results
Tolerance of Saccharomyces cerevisiae to Se: In the treatments used to de ne the dose of tolerance to Se, concentrations of 0 µg mL − 1 (T1), 30 µg mL − 1 (T2), 60 µg mL − 1 (T3), 120 µg mL − 1 (T4) and 240 µg mL − 1 (T5) of sodium selenite were added to the medium. The results obtained showed that the cultivation of yeasts under the conditions of the T1 (control), T2 and T3 treatments obtained colonies growth within 48 hours after inoculation. The composition of the T3 medium was the maximum Se concentration that yeasts managed to grow. On the other hand, no colonies growth was observed within 48 hours after incubation, on substrates subjected to treatments T4 and T5. In the T1 treatment (without the addition of sodium selenite) with 10 − 5 CFU mL − 1 dilution of the inoculum, the largest number of colonies was obtained per unit volume of substrate, with 3.9 x10 2 CFU mL − 1 . These colonies showed white color, circular shape, not rough and sizes varying between 0.8 and 5 mm in diameter. Under the conditions of treatments T2 and T3, smaller increases in the number of colonies were observed, with approximately 2.8 and 0.1x10 2 CFU mL − 1 , respectively. It was also identi ed that these colonies showed white color, circular shape and not rough. However, ranging from 2 and 5 mm in diameter. In the conditions of treatments T4 and T5, no growth of colonies was observed within 48 hours of incubation, as can be seen in Fig. 1, with the images of the treatments of tolerance.
As no colony growth was identi ed at a concentration of 120 µg mL − 1 (T4) in YEPDA culture medium, it was necessary to de ne the tolerance interval between 60 (T3) and 120 (T4) µg mL − 1 . However, at the concentration of 70 µg mL − 1 of sodium selenite (T6), there was no growth of colonies. Therefore, it was de ned that 60 µg mL − 1 (T3) was the maximum concentration with cell growth without the need for an adaptive process.
Adaptation of Saccharomyces cerevisiae to Se: The adaptive process carried out for 64 days in 32 consecutive cultivation cycles made expansion of tolerance capacity of yeasts to Se from 60 µg mL − 1 to up to 246 µg mL − 1 possible. As the concentration was increased, changes in the color of the yeast colonies were observed (Figs. 2, 3, 4

and 5).
At the beginning of the adaptation process, the colonies were light beige and shiny, as the doses of Se in the medium were increased. Then the yeast colonies began to show a darker color so that, after 32 cultivation cycles, the colonies had an intense reddish brown color. In addition, there was a reduction in the number of colonies and an increase in the roughness of the colonies on the entire surface, but mainly at the edges. Changes in the odor of the colonies were also observed, which began to show similarity to the smell of hydrogen sul de.
In Fig. 6a it is possible to clearly see the color change from beige to orange red, when the yeasts were grown at 96 µg mL − 1 medium. In Fig. 6b, colonies with intense orange red coloring and roughness at the edges were found when the yeasts were grown at 150 µg mL − 1 .
Although yeasts grow in concentrations from 222 to 246 µg mL − 1 , a reduction in the growth speed of colonies that started to develop after 24 or 36 hours of incubation was observed. While at concentrations of 60, 66 and 72 µg mL − 1 , yeast colonies could be observed in the rst 12 hours of incubation.
With the presence of high concentrations of Se in the substrate, morphological changes were observed in the yeast cells. At T6 (90 µg mL − 1 of sodium selenite in the culture medium) was possible to verify cell clusters, increase in size and transverse area of the cells, using an optical microscope (Fig. 7).
The changes promoted in the yeast cell wall were observed by means of scanning electron microscopy, through which it was possible to observe the wrinkling of the surface of the yeast cells and changes in the shape of the cells when they were subjected to concentrations of 60, 120 and 240 µg mL − 1 of sodium selenite (Fig. 8), once the characteristic of yeast without the presence of Se is smooth.

Discussion
The yeasts submitted to Treatment T2 (60 µg mL − 1 of sodium selenite) grew within the estimated time of 24 to 48 hours, without the need for cell adaptation of the yeasts to the Se; therefore, the maximum concentration of yeast (S. cerevisiae), strain Y904, supports without the adaptive process. Similar results were found in the works of Assunção (2011)  With the increase in the concentration of sodium selenite, the yeasts showed an intense reddish brown color. Changes in colony odor were also observed. It was similar to the smell of hydrogen sul de.
According to Suhajda et al. (2000), this may occur due to the substitution of Sulfur by Se in the enzymes. The Se compounds follow the same metabolic pathways and the metabolites are analogous to that of S. These changes in cell staining can be explained by the biotransformation that happens inside the cell, when the selenite (transparent coloring) is reduced to Se amorphous (reddish coloring) (KONETZKA 1977).
In the work of Bierla et al. (2013), the substitution and degree of substitution of sulfur for Se in methionine and cysteine, in Se-rich yeasts, using plasma mass spectrometry inductively coupled by capillary HPLC (ICP-MS), used in parallel to capHPLC-ESI-MS was investigated. As a result, substitution of cysteine sulfur is three times less frequent than that of methionine sulfur. Taking into account the amounts of methionine and cysteine available in yeast cells, they concluded that the estimate of selenocysteine concentration in Se-rich yeasts was 15 to 30% of selenomethionine. Birringer et al. (2002) demonstrated that the small amounts of organic Se compounds in plants, yeasts, bacteria or animals are isologists of sulfur compounds and the enzymes involved in the metabolism and trans-sulfurization pathway do not normally discriminate between sulfur and Se compounds. Despite being of the same species, S. cerevisiae, and having the ability to bioaccumulate several elements and, as a result, tolerate higher concentrations in the medium, the different strains show different behaviors to stress and speci c variations. There are studies with yeasts of the same species that tolerate different concentrations of Se (WHITE 1987). As in the results obtained by Wang et al. (2010), the addition of 90 µg mL − 1 in the late exponential development phase of yeast of lineage GS2, was the highest tolerated concentration taking into account the decrease in biomass production.
In the present work, the adaptive process allowed the yeast to tolerate up to 246 µg mL − 1 of sodium selenite in the culture medium. Due to the gradual increase of Se in the medium and the metabolic interactions caused by Se, the yeasts presented different characteristics, such as, the reduction of the cell multiplication speed, roughness, intense hydrogen sul de odor and increase in the intensity of the reddish brown color. Such results were similar to those cited in the literature. Suhajda et al. (2000), enriched S. cerevisiae also using sodium selenite, obtained a reddish color in the yeast cells.
The adaptive ability of yeast can be acquired when exposed to the appropriate selection pressure (WHITE 1987). According to Bronzetti et al. (2001), the addition of high concentrations of sodium selenite to the culture medium demonstrated to have a mutagenic effect in the yeast cells of the species S. cerevisiae D7, generating a 70% decrease in cell survival when compared to the control group.
The change in all these characteristics (change in color, roughness, odor, size and number of cells) with the increase in the concentration of sodium selenite, demonstrates how the yeasts have undergone modi cations and / or adaptations in order to survive the stress caused by the enrichment of the culture medium with Se, which may induce to believe that adaptive evolution of yeast cells has occurred.   Optical microscopy of yeasts grown in YEPD medium at 30˚C after 48 hours, with a 400x magni cation.