Two yeast strains, Saccharomyces cerevisiae ATCC 7090 and Rhodotorula glutinis CCY 20-2-26, were used in this study. The strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the Culture Collection of Yeasts (CCY, Bratislava, Slovakia). The strains were stored at 4 °C on solid yeast peptone dextrose (YPD) medium (BLT, Lodz, Poland).

The biomass yield of yeast cells was determined after centrifugation (Eppendorf 5810 Centrifuge, Hamburg, Germany) (3500 rpm; 10 min) of 30 mL of post-culture fluid in dry, pre-weighed 50 mL Falcon tubes. The supernatant was decanted for selenium content determination, and the sediment was dried (SML Zalmed Dryer, Warsaw, Poland) (80 °C) to a constant weight. Biomass yield was recalculated per 1 L of medium and expressed in grams of dry weight (g_d.w./L).

The spectrophotometric method used to determine selenium is based on measuring the absorbance (wavelength 546 nm) of the coloured compound Variamine Blue (VB, Sigma-Aldrich, Warsaw, Poland). The intensity of VB staining depends on the number of its molecules reacting, initially triggered by selenium ions [23]. The method involved taking 1 mL of the supernatant into 50 mL volumetric flasks containing 5 mL of a 2% KI solution (Merck, Warsaw, Poland) and 5 mL of 2 M HCl (Merck, Warsaw, Poland), followed by gentle mixing to release iodine. After iodine release, 2.5 mL of 0.05% VB reagent and 10 mL of 1M sodium acetate were added to each flask. The volume was then made up to 50 mL with distilled water, and the absorbance was measured (BIO-RAD SmartSpec 3000 spectrophotometer, Warsaw, Poland) at 546 nm against a control sample. The average absorbance results were converted to selenium content in the sample using the regression equation (y = 0.0527x – 0.009) (Fig. 1). The obtained value was corrected considering the culture volume. The difference between the initial and final selenium concentration in the medium indicated the amount of selenium accumulated by the yeast cells. The selenium content accumulated per gram of dry weight of the tested yeast strains was calculated based on the biomass yield.

The morphology of the studied microorganisms was observed using an optical microscope (OPTA-TECH, Warsaw, Poland). Measurements of yeast cell length and width were recorded using Opta View 7 software (OPTA-TECH, Warsaw, Poland).

The liquid yeast biomass (1.5 mL) was immersed in 2 mL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, Warsaw, Poland), to which 0.5 g of glass beads approximately 500 µm in diameter were added and mixed for 1 h at 70 rpm (Rotator MultiBio RS-24, Biosan, Riga, Latvia). Then, 2 mL of acetone (Sigma-Aldrich, Warsaw, Poland), 2 mL of petroleum ether (with 0.25% BHT)(Sigma-Aldrich, Warsaw, Poland) and 2 mL of 20% sodium chloride (Sigma-Aldrich, Warsaw, Poland) were added to each tube, and the samples were mixed again under the same conditions. To separate the phases, the tubes were centrifuged at 20 °C at 3000 rpm for 5 minutes (Eppendorf 5810 Centrifuge, Hamburg, Germany). The separated ether phase containing the carotenoids was transferred to a cuvette, and the absorbance was measured at $\lambda$ = 490 nm using a spectrophotometer (Eppendorf BioSpectrometer, Hamburg, Germany). The ether fraction was transferred to clean tubes for further carotenoid profile analysis. If the biomass colour did not change after extraction, the procedure was repeated from the DMSO addition step. The carotenoid content per gram of dry substance was calculated using the formula: A_max $\times$ D $\times$ V/(E $\times$ W), where A_max represents absorbance at 490 nm, D is the sample dilution factor, V is the ether fraction volume, E is the extinction coefficient of carotenoids (0.16) and W is the dry weight of yeast (g_d.w.).

To identify the extracted carotenoids, after evaporating the petroleum ether under nitrogen atmosphere, 1.2 mL of the HPLC mixture consisting of acetonitrile, isopropanol and ethyl acetate in a ratio of 4:4:2 was added. The mixture was filtered through a 0.45 µm pore size nylon filter and analysed chromatographically on a C18 analytical column (Bionacom, 250 mm $\times$ 4.6 mm, 5 µm). The column was placed in a thermostat set at 25 °C, with a mobile phase flow rate of 0.7 mL/min. The detector wavelength was 490 nm. $\beta$-Carotene identification was based on the retention time of the standard, while torulene and torularhodin were identified based on the retention times of the standards separated using thin-layer chromatography (TLC). After the analysis, the areas of the respective chromatographic peaks were calculated to determine the percentage content of individual carotenoid fractions [24].

The analysis was conducted using the Kjeldahl method to determine the total protein content in the yeast cell biomass. Additionally, changes in protein content in the experimental media were assessed compared with the control sample of the studied yeast strains. One hundred mg of dry yeast cell substance was weighed and mineralized (Buchi Digestion Unit K-435, Germany) in 96% sulfuric acid (VI) in the presence of a catalyst (Kjeltabs CT/3.5 tablets, FOSS Analytical, Hillerød, Denmark). After the process was completed, the solution was cooled, then distilled (KjelFlex K-360, BÜCHI Labortechnik AG, Munich, Germany) and automatically titrated (TitroLine 5000, SI Analytics, SI Analytics GmbH, Mainz, Germany) using 0.1 M acid (Kjeltec 2100, Foos). The nitrogen content was converted to total protein content using the conversion factor 6.25.

To determine the lipid content in the dry yeast biomass, the Bligh and Dyer method was used [25]. To disintegrate the cell wall, 10 mL of 1M HCl was added to 200 mg of dry yeast biomass and incubated for 2 h at 60 °C (Water Bath SBS-TWB-2004, Steinberg Systems, Berlin, Germany). After cooling the samples, lipid extraction was performed using a chloroform (Sigma-Aldrich, Warsaw, Poland) and methanol (Sigma-Aldrich, Warsaw, Poland) mixture (1:1 ratio). After 20 minutes of intense shaking (Rotator MultiBio RS-24, Biosan, Riga, Latvia), 5 mL of a 20% sodium chloride solution (Sigma-Aldrich, Warsaw, Poland) was added to facilitate phase separation. The samples were then centrifuged at 3500 rpm for 10 min (Eppendorf 5810 Centrifuge, Hamburg, Germany). The chloroform phase was transferred to a pre-weighed tube. The chloroform was evaporated under a nitrogen atmosphere (Reacti-therm III #TS-18823, Thermo Fisher Scientific, Waltham, MA, USA), and the fat content was determined gravimetrically.

The obtained research results were analysed using analysis of variance in the Statistica 13.3 program (StatSoft, Warsaw, Poland). The significance of differences between the means in the respective groups was verified using Tukey’s honestly significant difference (HSD) test at a significance level of $\alpha$ = 0.05. Normal distribution was confirmed by conducting the Shapiro-Wilk test.

The growth of S. cerevisiae ATCC 7090, expressed as biomass yield, was dependent on the selenium concentration in the growth medium. After 24 h of cultivation in the control medium, this strain produced 9.67 g/L, and in subsequent days, the values were not statistically significant in the same control medium (Fig. 2). Adding selenium to the medium at a dose of 0.5 mg Se⁴⁺/L significantly reduced the biomass yield compared with the control to 6.32 g/L after 24 h of cultivation, and as in the control medium, its values did not change significantly in subsequent days. Similar biomass yield values were noted for selenium doses of 1, 2 and 5 mg Se⁴⁺/L. When selenium was added at 10 and 20 mg Se⁴⁺/L, the biomass yields after 24 h of cultivation were 5.67 and 4.57 g/L, respectively. The highest doses of selenium (30 and 40 mg Se⁴⁺/L) resulted in the greatest inhibition of yeast growth (2.88–3.70 g/L). Additionally, in cultures with selenium doses of 30 and 40 mg/L, the formation of a red precipitate (elemental selenium) was observed at the bottom of the flasks.

The growth results of R. glutinis CCY 20-2-26 differed from those obtained for S. cerevisiae ATCC 7090 (Fig. 3). After 24 h of cultivation, the biomass yield in the control medium without selenium reached 16.57 g/L. This is 71% higher than that obtained for S. cerevisiae ATCC 7090 after the same cultivation time. The biomass yield remained at a similar level throughout the cultivation process. The addition of selenium to the medium in amounts ranging from 0.5 to 5 mg Se⁴⁺/L did not significantly reduce biomass yields. The growth limitation compared with the control was noted for cultures conducted in media with 20, 30 and 40 mg Se⁴⁺/L, with the greatest limitation observed for the highest doses. After 72 h of cultivation in media with initial selenium concentrations of 20–40 mg Se⁴⁺/L, the biomass yields of R. glutinis CCY 20-2-26 ranged from 5.40 to 5.80 g/L. In cultures with the highest selenium doses, the yeast biomass lost its typical red colour. The presence of high selenium doses may cause changes in cellular metabolism. Enzymes and metabolic pathways responsible for pigment synthesis (desaturases) may be inhibited or altered by selenium, leading to reduced pigment production [26]. Selenium can directly react with pigments present in yeast cells. Pigments like carotenoids, which can give yeast a red colour, may undergo chemical modifications in the presence of high selenium concentrations, leading to changes in their colour properties. Similar results were observed by Kieliszek et al. [21] studying the effect of selenium on carotenoid production by the yeast R. mucilaginosa MK1, which reduced carotenoid concentrations in yeast cells.

The biomass yield results of the two studied yeast strains show significant differences in cell reactions to selenium-induced stress in the medium. Significant biomass yield reduction compared with the control in S. cerevisiae ATCC 7090 cultivation was caused by the smallest selenium doses, while in R. glutinis CCY 20-2-26, only selenium addition above 20 mg Se⁴⁺/L resulted in biomass yield reduction compared with the control at each time point. The results of this stage of the study demonstrated greater selenium tolerance in red yeast compared with the widely studied yeast S. cerevisiae ATCC 7090.

In the study conducted by Kieliszek et al. [23], the selenium binding ability of the biomass of S. cerevisiae ATCC MYA-2200 and C. utilis ATCC 9950 was assessed. Sodium selenite (IV) salts were used in concentrations of 10, 20, 40 and 60 mg Se⁴⁺/L. For S. cerevisiae, a dose of 10 mg Se⁴⁺/L after 24 h of cultivation significantly affected the biomass yield, reducing it from 9.80 g/L (control medium) to 6.7 g/L (a reduction of 46%). In this study, for S. cerevisiae ATCC 7090, this value decreased from 9.67 g/L (control) to 5.06 g/L (a reduction of 47.7%) under the same cultivation conditions. Comparing the results obtained in Kieliszek et al. [23] for C. utilis ATCC 9950, they resemble those obtained for R. glutinis CCY 20-2-26. After 24 h of cultivation without selenium, the biomass yield of Candida utilis ATCC 9950 was 16.5 g/L, the same as obtained in the control medium of R. glutinis CCY 20-2-26. Selenium supplementation at a dose of 10 mg Se⁴⁺/L reduced the yield of C. utilis ATCC 9950 to 15 g/L. The same dose for R. glutinisCCY 20-2-26 caused an identical biomass yield reduction.

Hyrslova et al. [27] conducted a study on the growth of seven different types of bacteria: Streptococcus, Lactococcus and Enterococcus, using various Na₂SeO₃ concentrations (5, 10, 30, 50 and 100 mg/L). The growth of all four Streptococcus thermophilus CCDM 144, 570, 561 and 129 strains decreased with increasing selenite concentrations. In contrast to streptococci, selenium presence did not significantly affect the growth of Enterococcus faecium CCDM 922A and Lactococcus lactis subsp. cremoris CCDM 72 and CCDM 73 strains. After 24 h of cultivation, all studied strains were able to convert Na₂SeO₃ to elemental selenium, manifested by a reddish colour, especially in environments with higher selenite concentrations of 10 mg/L or more (30, 50 and 100 mg/L). These results may indicate similar selenium tolerance in bacteria compared with the S. cerevisiae ATCC 7090 strain.

Yang et al. [28] examined the impact of selenium on biomass production in C. utilis SZU 07-01 yeast. They conducted cultivation in a bioreactor (pH 6.0; 30 °C; 20 h; 200 rpm), with a fermentation medium containing 30 g/L glucose, 8 g/L (NH₄)₂SO₄, 3 g/L KH₂PO₄ and 0.25 g/L MgSO₄. Selenium was added as sodium selenite (Na₂SeO₃) at a concentration of 15 mg Se⁴⁺/L in two variants: (I) by adding selenium at the start of cultivation and (II) by adding selenium after 15 h of cultivation, i.e., in the logarithmic growth phase. Biomass yield following control cultivation reached 13.2 g/L, whereas in variant I, it dropped by approximately 33% to 8.9 g/L. Adding selenium to the 15-h culture did not negatively affect yeast growth (13.1 g/L). It can be concluded that selenium toxicity to yeast cells strongly depends on the growth phase, with cells in the adaptation phase being more sensitive to this micronutrient.

The addition of selenium salts to culture media can affect yeast cells in many ways, leading to the changes in yield discussed above. Potential causes of these effects include selenium toxicity, alterations in yeast metabolism or cell structures and interactions between selenium and other media components, leading to complex effects on eukaryotic cells [29]. Excessive selenium can cause metabolic and structural disturbances in cells, negatively impacting their ability to grow and divide. Selenium can influence changes in yeast cell metabolism by disrupting the redox balance and affecting the expression of genes related to metabolism and growth, which in turn can lead to differences in biomass production and cell shape [30, 31]. The biomass of yeast cells cultivated in selenium-enriched media showed significant differences compared to the biomass from the control media. This phenomenon is likely due to toxic metabolites (hydrogen selenide H₂Se; selenodiol H₂SeO₂) produced during selenium transformation in yeast cells, including potentially toxic elemental red selenium (Se0). The generation of such substances can significantly disturb redox balance and lead to the oxidation of essential cellular components. Cultivating microorganisms in stressful environmental conditions can enhance the expression of genes responsible for carotenoid biosynthesis, as observed in the red yeast R. glutinis CCY 20-2-26 studied [32]. Selenium addition can affect cell structure by modifying the chemical composition of the cell wall or cellular organelles. This, in turn, can affect cell shape and their ability to maintain structural integrity. It is important to conduct thorough research to identify or attempt to identify the specific mechanisms and pathways by which selenium affects different types of yeast cells.

The presence of various elements in the culture medium can lead to changes in microorganism morphology. According to the literature [12, 33], the presence of selenites (IV) and selenates (VI) in the culture environment can cause changes in yeast cell sizes owing to adaptive processes to unfavourable environmental conditions. The results of cell length and width measurements are presented in Tables 1,2. Yeast cells of S. cerevisiae ATCC 7090 cultured in the control medium after 24 h had an average surface area of 30.66 µm², which slightly decreased to 26.36 and 25.42 µm² after two additional days of cultivation. Selenium doses from 0.5 to 10 mg/L did not significantly affect the dimensions of S. cerevisiae ATCC 7090 cells. However, adding 20 mg Se⁴⁺/L to the medium and cultivating for 72 h resulted in significant changes in the morphology of S. cerevisiae ATCC 7090 cells, increasing the surface area to 41.42 µm². Similar results were obtained for selenium doses of 30 and 40 mg Se⁴⁺/L after 72 h of cultivation.

Yeast cells of R. glutinis CCY 20-2-26 were smaller than those of S. cerevisiae ATCC 7090. In the control sample, the surface area of cells ranged from 26.23 to 27.22 µm² with no significant differences observed in each day of cultivation. Like S. cerevisiae ATCC 7090, selenium concentrations in the medium 20 mg Se⁴⁺/L did not cause significant morphological changes compared with the control sample in each tested time unit. An interesting dependency was observed in media with selenium equal to $>$20 mg Se⁴⁺/L – as the selenium concentration increased, the surface area of cultured cells decreased. Selenium doses of 20, 30 and 40 mg/L after 48 h of cultivation caused a significant reduction in surface area compared with the control results to 21.25, 16.83 and 15.4 µm² (Fig. 4).

It is worth noting that the tolerance level of cells to selenium ions may vary depending on the type or species of yeast, related to the potential induction of oxidative stress. When yeasts exhibit low tolerance to selenium in the environment, it can lead to changes in the cell wall structure, affecting the overall cell architecture. Research by Kieliszek et al. [12] showed that in the cultivation of C. utilis ATCC 9950 in media containing selenium ions, intracellular structures degraded. This resulted in loss of membrane integrity and cytoplasmic leakage, especially at a selenium concentration of 40 mg Se⁴⁺/L after 72 h of cultivation. A significant reduction in yeast cell size due to selenium presence at doses of 30 and 40 mg/L was observed in R. glutinis CCY 20-2-26 yeasts from the first day of cultivation and on each subsequent day. Based on a study by Kieliszek et al. [12], it can be assumed that yeast cell size increase results from selenium accumulation in cellular structures such as vacuoles. The gradual reduction in yeast cell size in the stationary phase can be explained by the progressive detoxification process of selenium in cellular organelles. This mechanism involves the gradual reduction of Se⁴⁺ to volatile hydrogen selenide (H₂Se) and selenium compounds bound to glutathione. Another possible detoxification method that may lead to yeast cell size reduction is the transport of selenium compounds first to the cytoplasm and then outside the cell. This process may involve transport proteins and Golgi apparatus vesicles [34]. A study by Rajashree and Muthukumar [35] demonstrated that S. cerevisiae NCYC 1026 yeast cells cultivated in selenium-enriched medium showed a size reduction of 0.88 µm compared with control cells not exposed to this element.

Based on the conducted studies, it can be assumed that S. cerevisiae ATCC 7090 and R. glutinis CCY 20-2-26 yeast strains respond differently to the presence of selenium in the environment. S. cerevisiae cells increased their surface area, probably owing to increased selenium compound accumulation in their intracellular structures. An opposite trend was observed for R. glutinis cells, probably owing to an enhanced detoxification system. Interestingly, literature results provide conflicting information about yeast cell morphology changes due to selenium ions in the culture medium, making this topic highly relevant [21, 35]. More research results on various yeast strains are needed.

Selenium accumulation in yeast cells depended on the selenium concentration in the culture medium. The selenium content in S. cerevisiae ATCC 7090 yeast biomass after the first day of cultivation was lowest in the medium with the minimum selenium dose (0.5 mg Se⁴⁺/L) compared with the other selenium doses, amounting to 0.021 mg Se⁴⁺/g (Fig. 5). Adding selenium to the medium at a dose of 10 mg Se⁴⁺/L resulted in yeast accumulating 1.88 mg Se⁴⁺/g. A significant increase in selenium content in S. cerevisiae ATCC 7090 cell biomass after the first 24 h of experimental cultivation was obtained in the medium with an initial selenium concentration of 20 mg/L, reaching 4.27 mg/g compared with media with selenium content 20 mg/L. Selenium content values for yeast cells grown in media with initial selenium doses of 30 and 40 mg/L were 8.56 and 10.49 mg/g. In subsequent days of the experiment, no drastic changes were observed in the obtained results. After 48 h of cultivation, the selenium dose of 10 mg/L resulted in a significantly higher selenium content (2.03 mg/g) compared with media with the smallest selenium doses (0.5 and 1 mg/L). The selenium concentration of 20 mg/L led to 4.69 mg/g accumulated selenium after 48 h of cultivation, and the value obtained on the next day (after 72 h) did not differ significantly. Initial selenium doses of 30 and 40 mg/L resulted in the highest selenium accumulation values in yeast biomass in each of the three tested time units. Summarizing the results for the S. cerevisiae ATCC 7090 yeast strain, it can be concluded that increasing selenium concentration in the medium does not translate into a proportional increase in selenium content in yeast biomass. A significant increase in bound selenium was observed after the first day of cultivation in the medium with an initial selenium concentration of 20 mg/L compared with media with selenium content 20 mg/L. Cultivation for the next 2 days did not result in significant changes in bound selenium compared with the results from the first 24 h, and increasing the selenium dose by 50% (30 mg/L) and 100% (40 mg/L) compared with the 20 mg/L dose caused the formation of red precipitate after the first 24 h and each subsequent day of cultivation. The red precipitate indicates the presence of elemental selenium, and the formation of such substances can cause severe redox balance disturbances and the oxidation of important cellular elements [21].

For the R. glutinis CCY 20-2-26 yeast strain, after the first 24 h of cultivation, selenium concentrations of 0.5–5 mg/L resulted in selenium accumulation in the biomass of the red yeast at a level of 0.01–0.29 mg Se⁴⁺/g (Fig. 6). An initial selenium concentration of 10 mg/L led to the binding of this element by the yeast cells in the amount of 0.64 mg/g, and doubling the selenium addition to the experimental medium significantly increased selenium accumulation (compared with the 10 mg/L dose) to 1.4 mg/g (for 20 mg/L). Further increases in selenium concentration in the medium resulted in a proportional and significant increase of this element in the yeast cell biomass compared with the results for media with selenium content 20 mg/L. In the medium supplemented with selenium at a dose of 30 mg/L, selenium accumulation reached 3.48 mg/g, while in the medium with a concentration of 40 mg/L, 5.03 mg/g was obtained. After 48 h of cultivation, no significant changes in selenium accumulation by R. glutinis CCY 20-2-26 cells were observed for concentrations ranging from 0.5 to 20 mg/L. An increase in bound selenium in the yeast biomass was observed with an initial concentration of this element at 30 and 40 mg/L, characterized by results of 5.38 and 7.53 mg/g, respectively. On the third day of cultivation (72 h), the values of accumulated selenium by the yeast cells resembled those obtained after 48 h of cultivation.

Comparing the results obtained for the two tested strains, it can be observed that the Rhodotorula yeast showed just over half selenium accumulation but exhibited better tolerance to higher selenium doses owing to twice the biomass yield compared with the results for S. cerevisiae ATCC 7090.

Several factors influence the ability of yeast cells to accumulate selenium – cultivation conditions, initial selenium concentration in the experimental medium and the type of microorganism studied [10]. In the study by Martiniano et al. [36], analyses was conducted using two different strains of S. cerevisiae (Lalvin ICV D47, 174, 193 and 405). A selenium dose of 15 mg Se⁴⁺/L resulted in the accumulation of this element at levels of 0.66 and 1.1 mg/g, respectively. Other two strains of S. cerevisiae (EC1118 and AW106) in the study by Yoshinaga et al. [37] showed binding of 3.1 and 3.5 mg Se/g, respectively, in a medium with an initial selenium dose of 34.58 mg/L. The study conducted by González-Salitre et al. [10] on the S. boulardii strain demonstrated selenium accumulation of 0.5, 0.79 and 3.4 mg Se/g after 3, 6 and 9 h of cultivation, respectively, with an initial selenium concentration in the medium of 74 mg/L. In the study conducted by Kieliszek et al. [23], the best selenium accumulation results were achieved in the cultivation of the S. cerevisiae ATCC MYA-2200 strain, with 1.61 mg Se⁴⁺/g obtained after adding 10 mg Se⁴⁺/L over a 72-h period. In the case of the C. utilis ATCC 9950 strain, 1.84 mg Se⁴⁺/g was obtained after adding 20 mg Se⁴⁺/L over 72 h. Analysing the efficiency of selenium utilization, yield and biomass content, the optimal selenium amounts were evaluated at approximately 15 mg Se⁴⁺/L for S. cerevisiae and 25 mg Se⁴⁺/L for C. utilis. The optimal cultivation time in both cases was 48 h. The study by Nam et al. [38] showed that the addition of selenium to the YM culture medium at concentrations ranging from 30 to 125 mg Se⁴⁺/L resulted in an increase in selenium content in the cell biomass of S. cerevisiae 6M from 1.04 to 5 mg/g. The study on R. glutinis yeast provided results confirming selenium accumulation at a level of 5.3 mg/g [13]. The study by Kieliszek et al. [21] reported new information on selenium accumulation by red yeast. The highest efficiency in selenium absorption from the environment was observed on the fourth day of cultivation, where statistically significant differences between doses influenced the amount of this element in the biomass of the tested yeast strain, especially in the case of the maximum selenium dose (20 mg Se⁴⁺/L). As a result, after four days of cultivation, the yeast showed gradual selenium utilization efficiency from the experimental environment in the following proportions: 78.04%, 53.48% and 57.50% at selenium concentrations of 10, 15 and 20 mg Se⁴⁺/L in the medium, respectively [21].

Krausova et al. [39] investigated the selenium bioaccumulation capacity of commercial lactic acid bacteria strains Streptococcus thermophilus CCDM 144 and Enterococcus faecium CCDM 922A in the presence of various sodium selenite concentrations (0–50 mg/L). The researchers noted that the ability of lactic acid bacteria to accumulate selenium increased with increasing selenium concentration in the culture medium. Both strains showed the highest selenium accumulation using an initial dose of this element at 50 mg/L–7.3 and 6.5 mg/g for CCDM 144 and CCDM 922A strains, respectively. Pieniz et al. [40] also reported high selenium accumulation by the Enterococcus durans LAB18s strain. Enterococcus durans was cultivated in BHI medium (35 °C, 24 h, anaerobic conditions) containing selenium in the form of sodium selenite at concentrations of 15, 30, 60 and 120 mg/L. The studies showed a significant correlation between selenium bioaccumulation in the biomass and the concentration of this element in the culture medium. Selenium concentration in bacterial biomass increased from 8.4 to 61.5 mg/g with increasing sodium selenite concentration added to the medium, ranging from 15 to 120 mg/L. The rate of selenium bioaccumulation reached 5.68% at a sodium selenite concentration of 60 mg/L. The analysis of the percentage of selenium binding suggests that the degree of bioaccumulation of this element increases until the sodium selenite concentration reaches 30 mg/L.

Hyrslova et al. [27] evaluated the ability of various bacterial strains from the genera Streptococcus, Lactococcus and Enterococcus to accumulate selenium in a medium containing 10 mg/L and 50 mg/L Na₂SeO₃. At a concentration of 10 mg Se⁴⁺/L in the culture medium, selenium content ranging from 2.5 to 7.4 mg/g was observed in bacterial cells. Only the Streptococcus thermophilus CCDM 561 strain had significantly higher selenium content, reaching approximately 45 mg/g. As in the above example, higher selenium concentrations (50 mg/L) in the culture medium resulted in a 4.5 to 7.8-fold increase in total selenium content in bacterial cells (17–201 mg Se⁴⁺/g).

Ponce et al. [41] conducted the study on various methods of selenium enrichment in S. cerevisiae yeast. They found that the best approach is to use lower concentrations of Na₂SeO₃, ranging from 10 to 50 mg/L, in the early logarithmic growth phase of the yeast. Additionally, temperature and pH are important factors influencing the selenium enrichment process. Yin et al. [42] demonstrated that the optimal conditions for selenium enrichment of S. cerevisiae are a temperature of 27 °C and a pH environment of 5.8. The culture was conducted in PDB (Potato, dextrose, broth) medium at 160 rpm. The study by Khakpour et al. [43] found that for the biomass of baker’s yeast S. cerevisiae PTCC 5010, the highest selenium binding level was 12.5 mg Se⁴⁺/g at a pH of 5.0. Kieliszek et al. [33] reported that pH in the range of 4.0–6.0 does not significantly affect selenium binding by yeast cells, hence the diversity of starting pH in different researchers’ studies, and in this study, the pH of the experimental medium was set at 4.8, which falls within the above range.

Differences in selenium accumulation by the same species of yeast may be due to metabolic variability [10]. Phosphate transporters and the regulation of their activity through phosphate concentrations are another factor influencing the interaction between selenium ion resistance and its accumulation. It has been shown that conditions characterized by low phosphate levels in the culture medium increase the activity of low-affinity phosphate transporters, leading to increased selenium uptake by yeast cells [14, 37].

One of the yet uncharacterized compounds is elemental selenium (Se⁰). There are reports in the available literature about the synthesis of selenium nanoparticles (SeNP) by microorganisms through enzymatic reduction of Se^2- to Se⁰ [2, 10]. Yeasts absorb selenium from the medium and transport it through the membrane, where it reacts with glutathione (GSH) to form selenodiglutathione (GSSeSG). GSH continues the reduction process, converting it into selenenyl sulphide glutathione (GSSeH). In the next step, this selenenyl sulphide glutathione (GSSeH) undergoes spontaneous dismutation, leading to the formation of elemental selenium (Se0) and the regeneration of GSH [44].

Elemental selenium production likely occurs when yeasts are unable to biotransform selenium into organic compounds (selenoamino acids) owing to excessively high concentrations of this element in the culture medium. It has been observed that selenium concentration in the medium is directly proportional to the aggregation of elemental selenium, although this mechanism has not been thoroughly studied and remains partially unclear, associated with the Ostwald mechanism where smaller particles coalesce into larger aggregates without control over their size due to significant surface energy [45]. In the study by González-Salitre et al. [10] on S. boulardii, an initial selenium concentration of 74 mg/L resulted in the production of elemental selenium. For the S. cerevisiae ATCC 7090 yeast examined in this study, the threshold dose causing Se⁰ production is 30 mg/L. According to the study by Kieliszek et al. [23], using selenium concentrations exceeding 40 mg Se⁴⁺/L in experiments is uneconomical because a significant amount of selenium remains unused in the culture fluid. Under such conditions, a red precipitate can be observed, suggesting the presence of large amounts of reduced selenium. Biomass obtained under such conditions contains increased amounts of elemental selenium, which is characterized by low bioavailability [44].

This study concludes that the optimal conditions for producing selenium-enriched yeast from the S. cerevisiae ATCC 7090 strain involve culturing for 24 h in a medium with an initial selenium dose of 20 mg/L. Under such conditions, the yeast can bind 4.27 mg/g, and this amount of accumulated element, considering the literature, falls within the upper range of results. The upper intake limits for selenium in humans are not directly regulated at the European level. Relevant organizations, such as the WHO (World Health Organization) and EFSA (European Food Safety Authority), recommend a daily selenium intake for adults of 30–40 µg. The Food and Nutrition Board of the National Academy of Sciences notes that daily selenium requirements vary by age, being 40–70 µg for men and 45–55 µg for women [8, 46]. Proper preparation of selenium-enriched yeast biomass could easily help mitigate selenium deficiencies worldwide. Culturing for 24 h allows for additional economic benefits compared with cultures conducted for 48 h or more. Higher selenium doses lead to the precipitation of elemental selenium, and a longer culturing time does not significantly impact the selenium accumulation results. According to Kieliszek et al. [23], using selenium at concentrations above 40 mg Se⁴⁺/L in experimental conditions is uneconomical because a high level of selenium remains unused in the culture medium. Under these conditions, a red precipitate can be observed, suggesting the presence of large amounts of reduced selenium. Selenium in this form is biologically inactive due to its low bioavailability.

For the red yeast R. glutinis CCY 20-2-26, optimal selenium binding conditions were obtained by culturing for 48 h in a medium with an initial selenium ion content of 40 mg/L. This yeast strain proved to be more resistant to high selenium doses, and at the highest studied selenium concentration, it accumulated 7.53 mg Se⁴⁺/g. Considering the literature used to write the discussion of this part of the study, this result is record-breaking and is 42% higher than the result (5.3 mg/g) obtained by Wang et al. [13]. Such a high selenium binding result by the biomass and greater tolerance to high selenium doses compared with the S. cerevisiae strain may be related to the natural ability to produce carotenoid pigments.

Owing to the documented health benefits of carotenoids (combating neurological diseases, peptic ulcers, cancer), scientists are continually seeking new ways to obtain them [47]. Many carotenoids are currently produced through chemical synthesis, but as demand for natural colourants grows, production via microorganisms is gaining significance. Among microorganisms capable of producing carotenoids, yeasts of the genus Rhodotorula are particularly interesting. These organisms primarily synthesize $\beta$-carotene, torulene and torularhodin [26]. Despite abundant information on carotenoid production by Rhodotorula yeasts, the relationship between selenium accumulation capability and carotenoid content is poorly studied.

The carotenoid content in R. glutinis yeast biomass during cultivation in a control medium without selenium addition was the highest (Fig. 7). After 24 h of cultivation, the carotenoid content in the control sample was 163.92 µg/g, and after 48 and 72 h, it significantly increased to 205.07 and 200.14 µg/g, respectively. The addition of the lowest selenium dose (0.5 mg Se⁴⁺/L) after 24 h of cultivation caused a significant decrease in carotenoid content in the biomass (130.54 µg/g), and subsequent days of cultivation did not significantly affect their content in the biomass. The addition of selenium to the medium in doses of 1 and 2 mg Se⁴⁺/L significantly reduced carotenoid content in the biomass to 75.32–85.24 µg/g compared with the control sample. Selenium in amounts above 5 mg Se⁴⁺/L also negatively impacted the biosynthesis of carotenoids, limiting their amount in the biomass to only 16.14–40.05 µg/g.

Carotenoids are a key group of low-molecular-weight antioxidants in the non-enzymatic process. Many microorganisms, such as bacteria, molds and yeasts, can produce various carotenoids, including gamma and beta-carotene, lycopene, torulene, torularhodin and astaxanthin [47]. It is particularly noteworthy that yeasts belonging to the genera Rhodotorula, Rhodosporidium, Sporobolomyces and Phaffia demonstrate an exceptional ability to produce carotenoids even under harsh conditions such as UV radiation, high temperature, or the presence of solvents or heavy metals. The study by Bogacz-Radomska and Harasym [48] showed that the presence of trace elements in the medium has a varied impact on the profile and the quantity of carotenoids synthesized by Rhodotorula graminis DBVPG 7021. For example, the presence of aluminium (III) and zinc (II) stimulated the synthesis of beta-carotene and gamma-carotene, while zinc (II) and manganese (II) inhibited the production of torulene and torularhodin. Another study by Bhosale and Gadre [49] demonstrated that divalent cations such as zinc (II), cobalt (II), iron (II) and copper (II) had a stimulating effect on the production and accumulation of carotenoids in R. glutinis CCY 20-2-26 yeast cells.

Research conducted by Nechay et al. [50] on Phaffia rhodozyma yeast showed that as selenium concentration in the medium increased, the quantity of carotenoids in the yeast biomass proportionally decreased. During these studies, various doses of selenium in the form of sodium selenite were used, including 10 mg/L. The use of selenium at a concentration of 10 mg/L resulted in a two-fold reduction in carotenoid production compared with the control sample, from 160 to 85 µg/g (Fig. 8). For the studied R. glutinis CCY 20-2-26 strain, a selenium dose of 10 mg/L after 24 h of cultivation resulted in a reduction in carotenoid production compared with the control sample from 163.9 to 33.1 µg/g, after 48 h from 205.1 to 24.1 µg/g, and after 72 h of cultivation similar results were observed.

It was found that the presence of selenium in the medium in amounts $>$5 mg Se⁴⁺/L significantly reduces the total carotenoid content compared with the control sample and affects the percentage share of their fractions. An increase in the concentration of torularhodin in the medium with doses from 0.5 to 10 mg Se⁴⁺/L compared with the control sample (from 24.3% to 29 and 26.5%, respectively) was observed, while higher doses (20, 30 and 40 mg Se⁴⁺/L) decreased the chemical compound to 14 and 6.8%. A proportional increase in beta-carotene content was also observed with increasing selenium content in the medium compared with the control sample. The highest increase in beta-carotene was observed for the highest selenium dose (40 mg/L), which was 44.2%, compared with the control sample (30.8%). Beta-carotene is a powerful precursor of vitamin A, characterized by strong antioxidant properties [26].

The study by Díaz-Navarrete et al. [51] have shown that R. mucilaginosa 6S responds to the presence of selenium with a varied production of biomass, carotenoids, and other biochemical components. Selenium supplementation in the form of sodium selenite (Na₂SeO₃) showed that concentrations above 10 mg/L significantly limited biomass growth, which may be the result of selenium-induced oxidative stress. An increase in selenium concentration disrupted the oxidation-reduction balance of cells and led to lower carotenoid production. In the control sample, where yeast was cultivated without selenium supplementation, the carotenoid content was 221.3 µg/g of biomass. After introducing selenium at a dose of 30 mg/L at the beginning of the culture, the carotenoid content decreased to 63.3 µg/g of biomass, a decrease of about 71% compared to the control sample. When selenium was added after 48 hours, when the cells were already in the logarithmic phase, the carotenoid content was 163.3 µg/g biomass, which means a decrease of about 26% compared to the control. The obtained results indicate that adding selenium at the late stage of the logarithmic growth phase can promote the bioaccumulation of selenium while minimizing its negative impact on carotenoid biosynthesis.

Moreover, studies on the effect of selenium on carotenoid biosynthesis are scarce, so to complement the discussion, we cite the results of studies on other metal salts that may have a similar effect on producing these compounds in yeast. The addition of zinc ions to the medium significantly affects the biosynthesis of carotenoids by the yeast R. toruloides MH023518, especially at appropriate concentrations [52]. The study showed that using zinc oxide nanoparticles at a dose of 50 mg/L increased the total carotenoid production to 264 mg/L, while the control without the addition of ZnO reached only 124 mg/L. However, with a further increase in the ZnO concentration to 200 mg/L, the carotenoid content decreased to 152 mg/L. These results suggest that zinc supports carotenoid production, but only up to a certain level, above which the production of pigments is inhibited. Another study by Elfeky et al. [53] presents the results on the effect of aluminum ions on carotenoid production in R. glutinis (AS 2.703). The culture medium was supplemented with aluminum sulfate Al₂(SO₄)₃ at a concentration of 0.7 mM, which resulted in an increased total carotenoid content of 2.21 mg/L. Increasing the dose of aluminum sulfate resulted in a decrease in carotenoid production. The study’s results indicate that selenium may be an essential factor modulating carotenoid production, but its presence at too high concentrations inhibits this production. This finding is crucial for further studies on optimizing the cultivation conditions of yeasts of the genus Rhodotorula to obtain biomass rich in carotenoids. The results suggest that to increase the efficiency of carotenoid biosynthesis by yeast, it is essential to precisely adjust the selenium concentration in the cultivation environment, which may be the basis for new bioproduction strategies [51].

The study provided evidence that selenium negatively affects the concentration of carotenoid pigments in the analysed Rhodotorula yeast. The type, oxidation state and amount of this element present in the culture medium can influence the regulation of carotenoid enzyme activities, especially specific desaturases responsible for carotenoid biosynthesis. This enzymatic regulation occurs in red yeast cells, according to the works of Kot et al. [26].

Combining studies on selenium accumulation assessment and changes in carotenoid content in yeast cell biomass is a significant gap in the available literature. The selection of appropriate cultivation parameters and selenium concentrations in the starting medium can contribute to optimal selenium binding in yeast biomass while increasing desired carotenoid fractions (e.g., astaxanthin). Such advancement would undoubtedly represent a significant step in the discussed research field.

Microorganisms can synthesize various valuable compounds, such as lipids (especially single-cell oil, SCO). Oleaginous microorganisms are characterized by their ability to produce and accumulate over 20% lipids in dry cell mass. Oleaginous yeasts are rare among all yeast species, constituting only about 5% of the total population. The genera of oleaginous yeasts that have attracted the most attention from researchers are Trichosporon, Cryptococcus, Rhodotorula, Rhodosporidium, Candida, Yarrowia and Lipomyces [54]. The fat content in their biomass can reach up to 60% (Yarrowia lipolytica) [55]. Yeasts mainly produce palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (C18:3) [26].

After 24 h of cultivation, it was observed that the studied yeasts, S. cerevisiae and R. glutinis, showed no significant signs of fat production. The results of the quantitative analysis of extracted lipids (2.5–4.9% for S. cerevisiae (Fig. 9) and 2–3% for R. glutinis (Fig. 10)) suggest that these amounts are so small that they are not likely to be accumulated as storage substances. Rather, these lipids are present in cell structures, such as the cell wall-membrane complex. To better understand this issue, it is worth mentioning that cellular fats can be stored in various organelles within the cell. The main locations where intracellular lipids can be present are primarily cell membranes, where phospholipids form an essential part of the structure. The fats in yeast cells can also include lipids of the internal membranes of organelles, such as mitochondria, where they play important metabolic functions (beta-oxidation, lipid synthesis, apoptosis regulation) [56].

The study by Tahmasebi et al. [54] on the simultaneous production of carotenoids and lipids by Rhodosporidium babjevae yeasts showed that with a glucose content of 60 g/L in the medium, 10.5% lipids were detected, while the highest carotenoid concentration (312 µg/g) was observed at a glucose content of 10 g/L. A summary of lipid production by other yeast strains in the available literature is as follows: Rhodotorula mucilaginosa CCT 7688 (no lipid production capability) [57], Yarrowia lipolytica Po1g (up to 58.5%) [58], Rhodosporidium toruloides 5149 (up to 30.3%) [59], Yarrowia lipolytica 5054 (up to 29.3%) [59], Rhodosporidium toruloides RP (up to 59.8%) [60].

In conclusion, selenium does not affect the fat accumulation process in the microorganisms, indicating that this element has no significant impact on lipid biosynthesis in yeast cells.

Environmental condition changes during cultivation can affect gene expression related to the production of new proteins in yeast cells. An example of such changes is the addition of selenium to the cultivation medium. The study indicates [44] that the expression of selenoproteins in organisms reaches its maximum at moderate selenium concentrations. However, higher doses of selenium can lead to lipid peroxidation in cell membranes. This underscores the importance of precise control of microbial cultivation conditions to optimize the production of desired proteins and minimize potential negative effects of this biotechnological process.

In this study, the impact of increasing selenium doses on changes in protein content in the cellular biomass of tested yeast strains after 24 and 48 h of cultivation was examined (Figs. 11,12). The biomass of S. cerevisiae ATCC 7090 in the control sample contained 59.65% total proteins after 24 h of cultivation, and 52.23% after 48 h. A selenium dose of 10 mg/L caused a significant increase in proteins after 24 h compared with the control sample (69%); however, after another day of cultivation, this value returned to the level observed in the control sample after 24 h of cultivation. Selenium doses of 20, 30 and 40 mg/L did not cause significant changes in total protein content compared with the medium with an initial selenium concentration of 10 mg/L.

The biomass of R. glutinis CCY 20-2-26 yeast cells in the control medium had a total protein content of 55.02% after 24 h of cultivation, and 53.58% after 48 h. Selenium doses of 10 mg/L did not significantly affect the protein content compared with the control sample in the examined biomass. A concentration of 30 mg/L caused a significant increase in proteins in the red yeast biomass to 65%, but their content decreased to 60.4% after 48 h of cultivation. Increasing the selenium content in the medium to 40 mg/L did not cause significant changes in protein content compared with the medium with a selenium dose of 30 mg/L.

The results showed an increase in protein content during the cultivation of Saccharomyces and Rhodotorula yeasts in selenium-containing media. The presence of selenium in the medium seems to stimulate the biotransformation of this element into its organic forms, such as selenomethionine, selenocysteine or selenomethylselenocysteine. This, in turn, can result in an increase in the number of proteins involved in these processes, including homocysteine synthase, methionine synthase and selenomethyltransferase [44].

In another study conducted by Kieliszek et al. [33], selenium supplementation of C. utilis ATCC 9500 and S. cerevisiae MYA-2200 cultures at a concentration of 20 mg Se⁴⁺/L contributed to increased levels of essential amino acids, including lysine, leucine and valine, compared with the those in the control culture. Parrondo et al. [61] in their studies showed that longer cultivation times of Kluyveromyces marxianus CECT 1123 yeast are associated with autolysis in the stationary growth phase, leading to a significant decrease in soluble protein content in cellular structures. For K. marxianus yeast, Aggelopoulos et al. [62] and Yadav et al. [63] report protein content ranges from 43 to 59%. In contrast, Agboola et al. [64] report that the protein content in Saccharomyces cerevisiae and Cyberlindnera jadinii yeast is 50.1% and 46.3%, respectively.

Guaragnella and Bettiga [65] described the effect of acetic acid as a stress factor suggesting an increase in the expression of genes involved in transcription and protein synthesis in the initial growth phase (first 24 h) and stimulation of protein metabolism in the later growth phase (48 h) because of S. cerevisiae yeast cells responding to stress caused by the addition of acetic acid (1.2–12 g/L). According to Yuan et al. [29], in response to various stress conditions (thermal, osmotic, metals, acids, salts, H₂O₂), S. cerevisiae yeast can adapt to unfavourable conditions by remodelling the cell wall and membrane, reprogramming gene expression and making metabolic adjustments (e.g., increasing antioxidant production, changing lipid profiles, producing protective substances). It is worth noting that according to the literature, the high protein content in dried yeast biomass typically ranges from 35% to 53% [33]. However, the studied yeast strains showed even higher protein content, up to 70% (S. cerevisiae strain after 24 h in a medium with an initial selenium content of 40 mg/L) after selenium supplementation. These results suggest that selenium may affect yeast metabolic processes and can serve as a starting point for further research on the biochemical mechanisms of selenium’s impact on these organisms. Further research will allow for a better understanding of new metabolic pathways involving selenium or those it may intensify.