Potassium and Sodium Salt Stress Characterization in the Yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, and Rhodotorula toruloides

ABSTRACT

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is a vital cell factory, but more diverse cell factories are essential for the sustainable use of natural resources. Here, we benchmarked nonconventional yeasts Kluyveromyces marxianus and Rhodotorula toruloides against S. cerevisiae strains CEN.PK and W303 for their responses to potassium and sodium salt stress. We found an inverse relationship between the maximum growth rate and the median cell volume that was responsive to salt stress. The supplementation of K⁺ to CEN.PK cultures reduced Na⁺ toxicity and increased the specific growth rate 4-fold. The higher K⁺ and Na⁺ concentrations impaired ethanol and acetate metabolism in CEN.PK and acetate metabolism in W303. In R. toruloides cultures, these salt supplementations induced a trade-off between glucose utilization and cellular aggregate formation. Their combined use increased the beta-carotene yield by 60% compared with that of the reference. Neural network-based image analysis of exponential-phase cultures showed that the vacuole-to-cell volume ratio increased with increased cell volume for W303 and K. marxianus but not for CEN.PK and R. toruloides in response to salt stress. Our results provide insights into common salt stress responses in yeasts and will help design efficient bioprocesses.

IMPORTANCE Characterization of microbial cell factories under industrially relevant conditions is crucial for designing efficient bioprocesses. Salt stress, typical in industrial bioprocesses, impinges upon cell volume and affects productivity. This study presents an open-source neural network-based analysis method to evaluate volumetric changes using yeast optical microscopy images. It allows quantification of cell and vacuole volumes relevant to cellular physiology. On applying salt stress in yeasts, we found that the combined use of K⁺ and Na⁺ improves the cellular fitness of Saccharomyces cerevisiae strain CEN.PK and increases the beta-carotene productivity in Rhodotorula toruloides, a commercially important antioxidant and a valuable additive in foods.

INTRODUCTION

Microbial cell factories (MCFs) are critical drivers of biotechnology and are essential for the sustainable use of resources. The bioprocesses using MCFs can replace the unsustainable fossil fuel-based chemical processes (1, 2). In the industrial bioprocesses, an ideal MCF thrives under severe stress conditions such as hyperosmolarity, temperature, and pH and the inhibitory effects of substrate-product concentrations in the culture environment (3–5). In particular, the budding yeast Saccharomyces cerevisiae, one of the most commonly used MCFs, shows hypersensitivity toward salts (6). S. cerevisiae also exhibits a limited substrate-product spectrum that restricts its use in a broader range of bioprocesses. These limitations necessitate identifying more robust microorganisms with a broader substrate-product spectrum and benchmark those against the most common MCF for the development of bioprocesses. The present study pursues this goal by investigating sodium and potassium salt tolerances and interaction in nonconventional yeasts.

We used extensively characterized S. cerevisiae strains, CEN.PK 113-7D and W303, for benchmarking salt tolerance in nonconventional yeasts, namely, Kluyveromyces marxianus and Rhodotorula toruloides (7–13). These nonconventional yeasts show a broader substrate-product spectrum than S. cerevisiae. The nonconventional yeasts naturally consume both hexose and pentose sugars, abundant in renewable natural resources, and can produce specialty chemicals such as terpenoids, carotenoids, and biofuels (14–16). By impacting the water activity and increasing molecular crowding, higher concentrations of salts, nutrients, and metabolic by-products induce ionic, oxidative, and osmotic stresses in MCFs, affecting their performance (17–20). Three basic mechanisms protect cells against the adverse effects of higher solute concentrations, particularly cations, which are the present study’s focus. First, the molecular transporters regulate cations’ active diffusion at the cellular and organellar levels (21–25). Second, the intracellular mechanisms activate pathways, such as the high osmolarity glycerol (HOG) pathway, enhancing cellular fitness (26–28). Third, the sequestration of cations within vacuoles maintains intracellular and organellar pH for enzymatic reactions (29–31). These distinct mechanisms enable the efficient exchange of protons and maintenance of electrochemical gradients across the biological membranes (32, 33). The underlying difference in these mechanisms contributes to distinct salt stress responses in different cells and organisms, and an understanding of these differences is valuable for bioprocess development. Salt concentrations affect biochemical and biophysical properties at organellar and cellular levels, influencing their shape and volume. The quantification of biophysical properties such as volume provides a global readout for monitoring changes introduced by varying the salt concentrations (34, 35). A comparative description of cell and vacuole volume changes, in conventional and nonconventional yeasts, in response to salt stress is presently lacking in the literature. However, such a description will be valuable for designing efficient MCFs using these yeasts.

We developed a neural network-based method for optical image analysis and applied it to characterize potassium and sodium salt stress responses in yeasts. Our study provides a quantitative description of volumetric parameters for exponentially growing yeasts by analyzing more than one million data points in optical images (>820,000 cells and >290,000 vacuoles). Overall, the present study (i) investigated yeasts’ physiology and morphology in a chemically defined culture medium, (ii) examined the impact of different potassium and sodium salt concentrations on yeasts, (iii) evaluated the effects of potassium supplementation on sodium salt stress in yeasts, and (iv) demonstrated a proof-of-concept bioprocess application for the combined salt supplementation.

RESULTS

Characterization of yeasts in a chemically defined culture medium.

S. cerevisiae CEN.PK 113-7D (CEN.PK), S. cerevisiae W303 (W303), K. marxianus, and R. toruloides were cultivated in a chemically defined mineral medium containing glucose as the primary carbon source. The highest cation concentration in the reference culture medium was for K⁺ (0.025 M), followed by Mg²⁺ (0.002 M), and Na⁺ (0.6 × 10⁻⁵ M), and other cations were present in trace amounts (see Table S1A in the supplemental material). All exponentially growing yeasts showed distinct morphologies in this culture medium (Fig. 1A). The shake flask-grown cultures were used to determine growth and physiology differences among the studied yeasts. S. cerevisiae strains grew similarly in the shake flask cultures, where K. marxianus was the fastest and R. toruloides was the slowest growing yeast (Fig. 1B1). K. marxianus attained the highest cell density among yeasts indicating the highest biomass yield (Fig. 1B2). R. toruloides formed aggregates in the reference culture medium after 7 h of cultivation, making it infeasible to reliably ascertain its cell density past that time point and took more than six-times longer than S. cerevisiae to consume all the available glucose (Fig. 1B2; Table S1B). S. cerevisiae strains produced metabolic by-products such as ethanol, acetic acid, and glycerol but consumed those after the depletion of glucose (Fig. 1B2; Table S1B). Noticeably, CEN.PK produced two times (0.25 ± 0.01 g/liter) more acetic acid than the W303 strain (0.09 ± 0.0 g/liter) at the end of the glucose phase (12 h) in the reference culture medium (Table S1B). Among the nonconventional yeasts in the reference culture medium, the only observed by-product was ethanol produced by K. marxianus (Fig. 1B2; Table S1B).

FIG 1.

Characterization of yeasts in a chemically defined culture medium. (A) Representative morphology of exponentially growing cells. (B1) Yeasts’ specific growth rates (h⁻¹). All cultivations were performed in shake flasks. Mean values were calculated based on biological triplicate experiments. (B2) Yeasts’ physiology in the reference minimal medium using shake flasks. Data represent an average from triplicate experiments, and the detailed physiology profiles are available in Table S1B in the supplemental material. OD, optical density at 600 nm; AG, cell aggregates; GLU, glucose (g/liter); GLY, glycerol (g/liter); ACE, acetic acid (g/liter); EtOH, ethanol (g/liter). (C1) Cell volume (femtoliters [fL]) distribution at exponential growth phase showing the percentages of cells in different volume bins. The number of cells (n) used for the quantification was as follows: CEN.PK, 6,157; W303, 20,895; K. marxianus, 21,534; R. toruloides, 26,117. (C2) Cell volume distribution plot, indicating heterogeneity in the population of each yeast culture. Median cell volume (femtoliters, represented by symbols) of exponentially growing cells. Dashed lines represent standard deviations (SDs) and are calculated separately for each half of the nonnormal cell volume distribution, as represented in C1. For a representative visualization, both halves of SD values are plotted at 50% of the actual values. (D1) Vacuole volume (femtoliters) distribution at the exponential growth phase shows the percentages of vacuoles in different volume bins. The number of vacuoles (n) used for the quantification was as follows: CEN.PK, 1,825; W303, 14,634; K. marxianus, 15,412; R. toruloides, 11,777. (D2) Vacuole volume distribution plot, corresponding with the population heterogeneity. Median vacuole volume (femtoliters, represented by symbols) of exponentially growing cells. Dashed lines represent SDs as described for C2. (E) Median vacuole-to-cell volume ratios at exponential growth phase. The number of vacuoles and cells used to calculate this ratio are the same as for D1.

FIG 2.

Yeasts’ response to the supplementation of K⁺ in a chemically defined culture medium. (A) Relative specific growth rate, using a microplate reader, based on independent quadruplicate experiments. The reference culture medium’s specific growth rates (K⁺, 0.025 M; Na⁺, 0.6 × 10⁻⁶ M) were considered 100% for each yeast. Error bars indicate SDs. (B) Yeasts’ physiology in the reference minimal medium, K⁺ supplemented, in shake flasks. Data represent the averages from triplicate experiments, and detailed physiology profiles are provided in Table S1B and corresponding cellular morphology in Table S2B. Abbreviations are the same as used in Fig. 1. (C) Relative cell (n > 6,000) and vacuole (n > 1,800) volume distribution plots of exponential-phase cells, indicating changes in volumetric heterogeneity of the population. The first data point refers to the reference (REF). Log₂ fold change (log₂FC; median values, marked by symbols) are plotted relative to the reference condition. Dashed lines representing log₂FC (SD) are calculated separately for each half of the nonnormal cell and vacuole volume distributions. Log₂FC value was obtained by dividing the SD for the experimental condition with the reference value for each half of the nonnormal distribution. For a representative data visualization, both halves of the SD values are plotted at 50% of the actual values. Fig. S2B shows the percentage of bins of cell and vacuole distributions in response to the increased K⁺ concentration in the culture medium. *, P < 0.001 relative to the reference based on the Kruskal-Wallis test.

A neural network-based method was developed to quantify volumetric parameters using optical microscopy images (https://github.com/a-ill/Cell-Image-Analysis-Pipeline). The exponentially growing cultures were used for the analysis of images (>5,000 cells for each strain and condition) (Fig. 1C1 and C2). The vacuole volume was considered in our analysis since it is critical for controlling cell volume in the presence of salts (Fig. 1D1 and D2) (36). This analysis allowed the quantification of differences in distributions of cell and vacuole volumes in the exponential cultures (Fig. 1C1, C2, D1, and D2). A similar analysis method was previously used to show that the variability, using the median cell volume, in growth and cell division contributes to heterogeneity in the cultures (37). The stochasticity in the growth and cell division processes creates inherent noise in these parameters (37). The optical image analysis allowed us to quantify the vacuole and cell volume ratio (Fig. 1E). This ratio is considered crucial for controlling cell volume and aging in yeast (38, 39). Cell and vacuole volumes followed nonnormal distribution patterns (Fig. 1C1 and D1). For this reason, independent standard deviations for each half of the volume distribution were calculated (Fig. 1C2 and D2). The specific growth rate (h⁻¹) of each yeast was inversely proportional to the median cell volume, indicating smaller cell volume favored faster growth (Fig. 1B1 and C2). The reference yeast cell volume obtained in our analysis was in agreement with the previously reported cell volume for S. cerevisiae (40). One of our image analysis method’s advantages was counting the buds as potentially independent cells, which is otherwise a complex task requiring specialized techniques for counting mother and daughter cells (41). Therefore, various numbers of smaller cells, represented in our data (Fig. 1C1), were due to potential differences in yeasts’ budding patterns at the exponential growth phase. The relatively large vacuole volume likely contributed to the heterogeneity observed for W303 compared with that of other yeasts under the reference condition (Fig. 1C1, C2, D1, and D2). W303 and K. marxianus showed more prominent vacuole-to-cell volume ratios than CEN.PK and R. toruloides, indicating potentially distinct vacuolar functions among yeasts (Fig. 1E).

Effects of potassium salt supplementation in yeasts.

After benchmarking yeasts in the reference culture medium, a microplate reader setup was used to screen and identify a viable K⁺ concentration range for their characterization. For this screening, K⁺ concentration was varied from the reference level (0.025 M) to 2.0 M by using KCl (Fig. 2). All yeasts were viable up to a 1.5 M K⁺ concentration but did not survive at the next tested K⁺ concentration (2.0 M), except for R. toruloides (Fig. 2A). K. marxianus showed the highest sensitivity to an increase in K⁺ concentration by reducing specific growth rates by 25% at 1.0 M and 58% at 1.5 M (Fig. 2A). However, since K. marxianus’ reference specific growth rate was the highest among the studied yeast, it maintained the highest specific growth rate at viable K⁺ concentrations (Fig. 1B1 and 2A). K. marxianus was second only to CEN.PK in its sensitivity toward the K⁺ cation. CEN.PK showed 11% and 60% reductions in the specific growth rate compared with that of the reference at 1.0 M and 1.5 M concentrations, respectively (Fig. 1B1 and 2A). R. toruloides grew at a 54% reduced specific growth rate at a 2.0 M K⁺ concentration compared to that under the reference cultivation condition (Fig. 2A).

After the plate reader-based screening of the viable K⁺ concentration range, shake flask cultivations were used to determine yeasts’ physiological responses in the presence of a 1.0 M K⁺ concentration in the cultures where all the strains maintained 75% or more of the specific growth rate compared to that under the reference condition (Fig. 2B; Table S1B). S. cerevisiae strains responded differently to an increase in K⁺ concentration. In CEN.PK, glucose was entirely consumed, but it failed to consume the metabolic by-products produced during the growth (Fig. 2B; Table S1B). Interestingly, CEN.PK converted the produced ethanol to acetic acid, but the latter was not further metabolized (Fig. 2B; Table S1B). However, W303 consumed the metabolic by-products, except glycerol, which was finished only partially during the cultivation duration (Fig. 2B; Table S1B). Unlike CEN.PK, the W303 strain showed no impairment in ethanol or acetic acid consumption upon the increase in the K⁺ concentration (Table S1B). Because of these metabolic disparities, CEN.PK and W303 exhibited 65% and 37% lower cell density, respectively, than the reference cultures, where both strains showed similar cell densities (Fig. 2B; Table S1B). Again, K. marxianus showed the maximum cell density among cultivated yeasts despite the 33% decrease in cell density compared with that under the reference condition. K. marxianus completely consumed glucose and produced glycerol as the main by-product, which was also consumed by the end of cultivation (Fig. 2B; Table S1B). R. toruloides showed a significant trade-off between glucose consumption and cellular aggregate formation in the culture (Fig. 2B; Table S1B). The addition of 1.0 M KCl eliminated R. toruloides culture aggregates but led to a slower and partial glucose utilization (Fig. 2B; Table S1B). It also showed a small accumulation of glycerol, but no acetic acid or ethanol was detected in the culture broth (Fig. 2B; Table S1B).

The cellular and vacuolar volumes were calculated using exponential-phase shake flask cultures for the K⁺ concentration range (0.025 to 1.0 M) (Fig. 2C; see also Fig. S2A and B). For CEN.PK, heterogeneity in both median cellular and vacuolar volumes remained nearly constant, but the median volumes showed a significant change at higher K⁺ concentrations (Fig. 2C; Fig. S2A and B). In contrast, W303 showed the most heterogeneous population in the study, exhibiting a significant increase in median cellular and vacuolar volumes at the increased K⁺ concentrations (Fig. 2C; Fig. S2A and B). Though drastically different from each other, the vacuole-to-cell volume ratio slopes did not change significantly for S. cerevisiae strains with increased K⁺ concentration (Fig. S2B). K. marxianus showed an exponential decrease in median cell volume but a U-shaped pattern for median vacuolar volume, changing significantly with the increase in K⁺ concentration. For K. marxianus, the overall population-level heterogeneity for both cells and vacuoles initially appeared unchanged but increased at the highest concentration (Fig. 2C; Fig. S2A and B). For K. marxianus, vacuolar volume did not follow the decrease in cell volume with increased K⁺ concentration. Initially, K. marxianus vacuole volume decreased with the increase in K⁺ concentration, but at 1.0 M K⁺, it partially reverted toward the reference (Fig. 2C; Fig. S2A and B). The ascent in the vacuole-to-cell volume ratio slope with the increase in cell volume, which was present under the reference condition, disappeared at 0.3 to 0.6 M K⁺ concentrations, but a smaller angle than the reference reappeared at the 1.0 M K⁺ concentration. The slope increase was synergistic with vacuole volume (Fig. S2B). For R. toruloides, cellular and vacuolar median volumes significantly increased in similar directions, rendering the slope unaffected (Fig. 2C; Fig. S2B). R. toruloides showed the least imparted heterogeneity due to the increase in K⁺ concentration among the studied strains (Fig. 2C; Fig. S2A and B).

Effects of sodium salt supplementation in yeasts.

A viable Na⁺ concentration range for the four studied strains was identified using a microplate reader-based setup while maintaining the K⁺ concentration at the reference level (Fig. 3A). S. cerevisiae strains showed remarkably distinct responses to the increased Na⁺ concentration (Fig. 3A). The specific growth rate of CEN.PK was reduced by 75% at a 0.5 M Na⁺ concentration, whereas W303 grew at 59% reduced specific growth rate even at a 2.0 M Na⁺ concentration (Fig. 3A). This W303 growth contrasted with that at a 2.0 M K⁺ concentration, where both S. cerevisiae strains and K. marxianus were nonviable (Fig. 2A). In the specific growth rate comparisons, K. marxianus appeared similarly sensitive to K⁺ and Na⁺ cations, while R. toruloides was more sensitive to Na⁺ than to K⁺ cations (Fig. 2A and 3A).

FIG 3.

Yeasts’ response to the supplementation of Na⁺ in a chemically defined culture medium. (A) Relative specific growth rate, using a microplate reader, based on independent quadruplicate experiments. Yeasts’ specific growth rates in the reference medium (K⁺, 0.025 M; Na⁺, 0.6 × 10⁻⁶ M) were considered 100% for each yeast, and change in growth rates (mean values) are plotted relative to the reference. Error bars indicate SDs. (B) Yeasts’ physiology in the reference minimal medium, Na⁺ supplemented, in shake flasks. Data are averages from triplicate experiments, and detailed physiology profiles are available in Table S1B and corresponding cellular morphology in Fig. S3A. Abbreviations are the same as used in Fig. 1. (C) Relative cell (n > 6,000) and vacuole (n > 1,500) volume distributions of exponential-phase cells, indicating changes in volumetric heterogeneity of the population. The first data point is the reference (REF). Log₂ fold change (log₂FC; median values, marked by symbols) are plotted relative to the reference condition. Dashed lines representing log₂FC (SD) are calculated separately for each half of the nonnormal cell and vacuole volume distributions. Log₂FC value was obtained by dividing the SD for the experimental condition with the reference value for each half of the nonnormal distribution. For a representative data visualization, both halves of the SD values are plotted at 50% of the actual values. Figure S3B shows percentage bins of cell and vacuole distributions in response to Na⁺ supplementation in the minimal culture medium. *, P < 0.001 relative to the reference based on the Kruskal-Wallis test.

In a comparable setup to those of the K⁺ experiments, shake flask cultures supplemented with a 1.0 M Na⁺ concentration were used to determine yeasts’ physiological responses (Fig. 3B; Table S1B). Like the plate-reader screening experiments, S. cerevisiae strains responded differently to the increase in Na⁺ than to the increase in K⁺ concentration (Fig. 3A and 2A; Table S1B). In the presence of 1.0 M Na⁺ in shake flask cultures, CEN.PK and W303 showed only approximately 25% and 68% of the reference cell density, respectively. The cell density difference between 1.0 M Na⁺ and 1.0 M K⁺ supplemented cultures was 10% for CEN.PK (less in Na⁺) and approximately 5% for W303 (more in Na⁺) (Fig. 1B2, 2B, and 3B; Table S1B). CEN.PK showed even more residual metabolic by-products (33% ethanol, 35% glycerol, and 45% acetic acid) in the presence of 1.0 M Na⁺ than in the presence of 1.0 M K⁺ but similarly failed to consume those (Fig. 3B; Table S1B). In contrast to CEN.PK, W303 consumed ethanol and acetic acid but only partially consumed glycerol, showing an approximately 60% increase in the residual glycerol in the presence of 1.0 M Na⁺ compared with that for 1.0 M K⁺ at the end of cultivation (Fig. 3B; Table S1B). Still, the residual glycerol in W303 was less than that in CEN.PK (Fig. 3B). These metabolic disparities resulted in biomass differences in S. cerevisiae strains (Fig. 3B; Table S1B). Compared with that for S. cerevisiae strains, K. marxianus showed no accumulation of metabolic by-products and had the maximum cell density, approximately 10% less in Na⁺ than in K⁺ supplemented cultures, at the end of cultivation (Fig. 3B; Table S1B). K. marxianus did not show any glycerol production in the presence of Na⁺, unlike for K⁺, but ethanol metabolism appeared to be similarly affected by both cations (Fig. 3B and 2B; Table S1B). R. toruloides response was similar in the presence of both cations, except more glucose was consumed in the presence of Na⁺ than in the presence of K⁺ supplementation (Fig. 3B and 2B; Table S1B). The supplementation of both cations also prohibited the formation of aggregates in R. toruloides cultures (Fig. 3B; Table S1B).

The quantitative image analysis for cellular and vacuolar volumes was performed using exponential-phase yeasts from shake flask cultures, supplemented with a viable Na⁺ concentration range (0.6 × 10⁻⁵ to 1.0 M) (Fig. 3C; see also Fig. S3A and B). For CEN.PK, a parabolic pattern was observed for the median cellular and vacuolar volumes, although population heterogeneity remained nearly constant with the increase in Na⁺ concentration (Fig. 3C; Fig. S3A and B). Since CEN.PK cell and vacuole volume changes were in similar directions and proportions; their ratio’s slope did not change significantly (Fig. S3B). In W303, cell and vacuole volume changes occurred in opposing directions; their ratio’s slope increased with the increase in Na⁺ concentration (Fig. 3D; Fig. S3B). K. marxianus showed a significant decrease in the median cellular and vacuolar volumes with the increase in Na⁺ concentration but a relatively uniform heterogeneity in the population (Fig. 3C; Fig. S3A and B). The supplementation of both cations affected K. marxianus almost similarly (Fig. 2C and 3C). Noticeably, at median Na⁺ and K⁺ concentrations, K. marxianus’ vacuole volume appeared smaller than at the reference or the maximum studied concentrations (Fig. 2C and 3C). Some vacuolar fragmentation was also observed in K. marxianus at the maximum concentrations for both studied cations (Fig. S2A and S3A). In K. marxianus, the maximum slope value for vacuole-to-cell volume ratio was observed under the reference condition (Fig. 1A; Fig. S2B and S3B). The Na⁺ supplementation introduced a parabolic volume increase pattern in R. toruloides that was somewhat similar to that for CEN.PK (Fig. 3C). In R. toruloides, the vacuolar distribution patterns were similar in the presence of both studied cations, but cell volume changes were more prominent in response to Na⁺ than to K⁺ cations (Fig. 3C and 2C). R. toruloides showed the least heterogeneity among yeasts in the study, where the vacuole-to-cell volume ratio slope mostly remained unaffected by salt supplementations (Fig. S3B and S2B).

Modulation of sodium salt effects by supplementing potassium salt in yeasts.

The distinct responses to the supplementation of K⁺ and Na⁺ cations prompted us to investigate the yeasts’ response to their interactions in our study. In this instance, only shake flask cultivations were conducted at viable cationic concentration ranges, which were previously determined in the single cation supplementation experiments (Fig. 4A to C; see also Fig. S4A, B, and C and Table S1B). The different K⁺/Na⁺ ratios were obtained by changing the K⁺ concentration while maintaining Na⁺ at a 1.0 M concentration in the shake flask cultures (Fig. 4A to C).

FIG 4.

Yeasts’ response to K⁺ and Na⁺ supplementation in a chemically defined culture medium. All experiments were performed in the background of 1.0 M Na⁺ while varying K⁺ concentrations. (A) The specific growth rates for shake flask cultures are based on independent triplicate (for CEN.PK and W303) or duplicate (K. marxianus and R. toruloides) experiments. Error bars indicate SDs. Figure S4A shows changes in the lag phase and the final cell densities in response to dual cationic stress. (B) Yeasts’ physiology in the K⁺ and Na⁺ supplemented minimal medium in shake flasks. Data are averages from triplicate experiments, and detailed physiology profiles are available in Table S1B and corresponding cellular morphology in Fig. S4B. Abbreviations are the same as used in Fig. 1. (C) Relative cell (n > 7,000) and vacuole (n > 900) volume distributions at the exponential phase, indicating changes in volumetric heterogeneity of the population. The first data point is the reference (REF). Log₂ fold change (log₂FC; median values, indicated by symbols) is plotted relative to the reference. Dashed lines representing log₂FC (SD) were calculated separately for each half of the nonnormal cell and vacuole volume distributions. Log₂FC was obtained by dividing the SD for the experimental condition with the reference value for each half of the nonnormal distribution. For a Figure S4C shows percentage bins of cell and vacuole distributions in response to K⁺-Na⁺ supplementation in the minimal medium. *, P < 0.001 relative to the reference based on the Kruskal-Wallis test.

Interestingly, a 4-fold increase in the specific growth rate of CEN.PK was identified in the K⁺-Na⁺ interaction experiments (Fig. 4A). Two distinct cellular and vacuolar patterns were observed during this 4-fold increase in the specific growth rate, wherein volumes initially increased with the increase in K⁺ but reverted toward the reference levels at the maximum K⁺ supplementation (0.6 M) (Fig. 4A and C). This K⁺-Na⁺ supplemented volume and growth increase in CEN.PK was in contrast to an observed inverse relationship between cell volume and growth rate when different yeasts were cultivated under the reference culture condition (Fig. 1B1 and C2). However, the K⁺-Na⁺ supplementation induced a decrease in the W303 specific growth rate and caused an increase in cell and vacuole volumes (Fig. 4A and C). The vacuole-to-cell volume ratio slope did not significantly change for S. cerevisiae strains (Fig. S4C).

The K⁺-Na⁺ supplementation decreased the specific growth rate for K. marxianus, where, initially, cell and vacuole volumes increased, but at 0.4 M K⁺ supplementation, these volumetric parameters significantly decreased, with further decrease in the growth rate (Fig. 4A and C). The vacuole-to-cell volume ratio slope initially ascended with the increase in cell volume, but it decreased at 0.4 M K⁺ supplementation and was correlated with a significant decrease in vacuole volume (Fig. 4C; Fig. S4C). In the K⁺-Na⁺ supplementation experiments, K. marxianus showed a remarkable extension of the lag phase at a viable K⁺ concentration, and the supplementation of a 0.6 M K⁺ concentration was lethal (Fig. 4A; Fig. S4A and B). R. toruloides showed the most robust growth among investigated yeasts and maintained 82% of the reference specific growth rate, even at a maximum supplemented K⁺ concentration (0.6 M). Interestingly, R. toruloides maintained the specific growth rate but showed a significant decrease in cell volume, though the vacuole volume pattern was more complex (Fig. 4A and C; Fig. S4A and B).

In the K⁺-Na⁺ supplemented physiology data, the presence of dual cationic stress reduced cell density in yeasts compared with that for single cation concentrations (Fig. 4B). CEN.PK failed to consume ethanol and was unable to convert it to acetic acid, unlike in the presence of single cations (Fig. 4B; Table S1B). In CEN.PK, the K⁺-Na⁺ supplementation led to a >1.5-fold higher glycerol production, and consumption of other metabolic by-products was impaired (Fig. 4B; Table S1B). However, W303 consumed ethanol but accumulated two-times more glycerol and acetic acid than single cationic stress experiments and did not consume these metabolic by-products during the cultivation (Fig. 4B; Table S1B). Compared to S. cerevisiae strains, K. marxianus produced and consumed glycerol and acetic acid while completely exhausting available glucose, but its cell density was reduced by 43% in the K⁺-Na⁺ supplemented cultures compared with that in the single cation experiments (Fig. 4B; Table S1B). Under similar cultivation conditions, R. toruloides showed only a slight decrease in cell density compared with that for single cation supplementations. It accumulated three-times more glycerol while showing only partial glucose consumption, similar to K⁺-only supplementation, indicating the K⁺ cation negatively impacted glucose metabolism (Fig. 4B; Table S1B).

K⁺ modulates carotene production in Na⁺-supplemented R. toruloides cultures.

In bioprocesses, toxicity impacts titers and productivity (42). For the control of cellular toxicity and maintenance of a viable physiological environment, membrane transporters are vital. For example, the potassium antiporter-modulated K⁺/H⁺ electrochemical gradient across the membrane is proposed to be a general mechanism for enhancing tolerance to multiple alcohols when using S. cerevisiae in bioprocesses (42). The K⁺-Na⁺ supplementation provided an opportunity to evaluate the role of their symporters in bioprocess productivity. The availability of these cationic transporters differed in the studied yeasts (Fig. 5A; see also Table S5A and B). Our analysis found that R. toruloides lacked some of the organellar transporters, namely, Mdm38, Vnx1, and Vhc1, present in the other yeasts (Fig. 5A; Table S5A and B) (9, 12, 43, 44). As R. toruloides is a native producer of carotenes and previously showed increased production of carotenoids in the presence of osmotic stress (16, 28), the impact of K⁺-Na⁺ supplementation on carotene titers was evaluated in this study. Previously, it was established that maximum carotenoid production occurs during the stationary phase (16). Therefore, beta-carotene was measured at the stationary phase (after 96 h of cultivation), when R. toruloides cultures completely exhausted available glucose (Fig. 5B; Table S5C). The supplementation of 1.0 M K⁺ did not significantly improve carotene productivity (milligrams beta-carotene per gram glucose). Still, the supplemental 1.0 Na⁺ concentration induced a significant (P value <0.05) improvement in the beta-carotene productivity compared with the reference (Fig. 5B). Interestingly, the addition of 0.1 M K⁺ to 1.0 M Na⁺-containing cultures further enhanced the productivity of beta-carotene (P value <0.05) by 60% compared with the reference (Fig. 5B). However, a subsequent increase in K⁺ concentration reduced beta-carotene productivity, indicating a cationic stress tolerance limitation in R. toruloides (Fig. 5B). In glucose-grown R. toruloides cultures, cellular aggregate formation was observed in the reference culture medium but not in the presence of higher cationic concentrations. Therefore, a range of K⁺ and Na⁺ concentrations was tested to determine the minimal possible concentration that can effectively disallow cellular aggregate formation in the R. toruloides cultures. In this test, the supplementation of either 0.2 M KCl or 0.2 M NaCl to the reference cultures disallowed cellular aggregate formation without impacting the growth. The cell density and beta-carotene data for the reference were obtained in the presence of 0.2 M K⁺ concentration (Fig. 5B).

FIG 5.

Evaluation of K⁺ and Na⁺ supplementation on the production of beta-carotene in R. toruloides. (A) Yeasts’ cellular and organellar cationic transporters. The rectangles around the labels indicate the absence of cationic transporters, based on the currently available sequence comparisons, in R. toruloides. (B) R. toruloides culture cell density and the beta-carotene production yield on glucose (mg/g) in response to K⁺/Na⁺ cation supplementation. Data presented are from independent triplicate experiments. The beta-carotene measurements are based on the stationary-phase cultures (96 h), where glucose (10 g/liter) was entirely consumed. Error bars indicate SDs. *, P < 0.05 based on the Student’s t test with one tail, considering equal variance.

DISCUSSION

The present study investigated the impact of salt stress (KCl, NaCl, and their combinations) on conventional and nonconventional yeasts, describing their physiology and morphology. The potential application of K⁺-Na⁺ supplementation in cellular fitness and bioprocesses productivity is discussed. We showed that K⁺ modulates the toxic effects of Na⁺ cations, improving salt tolerance in CEN.PK and beta-carotene yield in R. toruloides. The supplementation of K⁺ is known to improve the tolerance for multiple stresses, including for alcohol in yeasts (42, 45, 46). For efficient cellular processes, such as protein synthesis, a high intracellular K⁺/Na⁺ ratio is essential in eukaryotes (19, 47). However, in industrial bioprocesses, Na⁺ is often present at a higher concentration, where it can be toxic to yeast because of its ability to readily replace K⁺ in essential cellular processes (3, 19).

Among investigated yeasts, some of the differences in stress tolerance pertain to the specific efflux and influx transporters (see Table S5A and B in the supplemental material) (25). The hypersensitivity of CEN.PK to Na⁺ but relative insensitivity to the same cation in W303 is attributable to the presence of an atypical plasma membrane ATPase related (PMR2) locus, renamed ENA for exitus natru or sodium exit (6, 21). The 4-fold increase in the specific growth rate of CEN.PK upon K⁺ supplementation in the presence of 1.0 M Na⁺ concentration is likely due to the differences in cation transporters among S. cerevisiae strains (Table S5A and B) (6). The role of monovalent cations in S. cerevisiae XT300.3A indicates the requirement of a certain K⁺ concentration (0.1 to 0.5 mM) for growth and suggests a modulation of growth upon the supplementation of Na⁺ (0.0 to 150 mM) or Rb⁺ (0.0 to 30.0 mM) cations (48). The range of investigated cations indicated in the parentheses is much lower than in the present study, but our results indicate a similar conclusion concerning the growth of CEN.PK. The reason for such growth modulation appears to be the role of intracellular K⁺ in counteracting the toxicity caused by Na⁺ at a higher concentration (49).

Though most transporter proteins consisted of the same amino acid sequences between W303 and CEN.PK strains, K⁺ importers (Trk1p and Trk2p) showed distinct peptide compositions (Table S5A and B). An atomic-scale model of these K⁺ importers in S. cerevisiae BY4741, which is the parental strain of CEN.PK and W303, showed that the amino acid specificity is important for the function and essential for correct positioning in the membrane (50). Interestingly among the nonconventional yeasts, K. marxianus showed only a single copy of the Trk family transporter, while R. toruloides possessed both Trk1p and Trk2p (Table S5A and B). K. marxianus and R. toruloides also possessed additional cationic transporters, namely, the Hak family transporters, compared with those possessed by S. cerevisiae (Table S5A and B). Besides, R. toruloides also contained cationic transporters of the Acu family and consisted of twice the number of main plasma membrane K⁺ and Na⁺ exporters, namely, Tok1p and Nha1p, compared with that in other yeasts (Table S5A and B).

R. toruloides showed the least amount of population heterogeneity, which may partially be due to its vacuole properties. Vacuoles help in osmotic regulation by importing excess ions into the vacuolar compartment and play an important role in several other biological functions such as intracellular pH regulation and autophagy (25). However, in our analysis, R. toruloides did not show vacuole transporters such as Vhc1p (K⁺ and Cl⁻) and Vnx1p (Na⁺/K⁺), which may explain its small vacuole volume. The mitochondrial K⁺ transporter Mdm38p, comparable to that in other yeasts, was also not detected in R. toruloides, but the analysis indicated an additional copy of the K⁺ transporter in the Golgi complex (Table S5A and B) (25). These transporter attributes may contribute to a relative population homogeneity and salt tolerance in R. toruloides, making it an attractive candidate for deploying as an MCF.

The lipid biosynthesis improves the tolerance for multiple stresses in yeasts by supporting membrane integrity. R. toruloides is an oleaginous yeast and produces more lipids in osmotic and oxidative stress environments (16, 51). Nitrogen and phosphate limitations are typically used for increasing lipid production; however, recently, salt stress was also indicated to increase lipid accumulation (28, 52, 53). In yeasts, triacylglycerols and steryl esters, both important for biofuels, accumulate in lipid droplets and can be turned over in a vacuole by a distinct autophagy process (52, 54, 55). Though lipid quantification was not the present study’s focus, their accumulation tends to be concomitant with beta-carotene, which increased by 60% upon combined K⁺-Na⁺ supplementation in R. toruloides cultures (Fig. 5B) (28). Interestingly, the increase in either K⁺ or Na⁺ concentration eliminated the formation of cellular aggregates in R. toruloides, which it uses in the native environment to adhere to plant surfaces (56). The presence of alkali-soluble materials such as mannose residues on the cellular membrane is instructive for the formation of these cellular aggregates (56). The K⁺ or Na⁺ supplementation likely disrupted such residue formation on the cell surface, making R. toruloides cultures free of aggregates.

Salt stress is a causative factor for the biosynthesis of osmoprotectants, such as glycerol, which increases osmotic tolerance (26). S. cerevisiae and R. toruloides compared with K. marxianus showed differences in glycerol production and consumption. K. marxianus produced much less glycerol than other yeasts in the study and consumed it completely, unlike other yeasts in the presence of higher salt concentrations. K. marxianus harbors the evolutionarily conserved HOG pathway and produces much more glycerol using lactose rather than glucose as a carbon source (27). Although we lack a direct comparison, these observations suggest that HOG pathway regulation and glycerol metabolism in K. marxianus may also be carbon source dependent as in S. cerevisiae (57). The NaCl-induced osmotic stress imparts a carbon source dependency in S. cerevisiae when cultivated on glucose and ethanol (57).

S. cerevisiae typically produces metabolic by-products such as ethanol and acetic acid on glucose and consumes those during the diauxic shift (58). However, the higher K⁺/Na⁺ concentrations impinged on ethanol and acetic acid utilization in CEN.PK. W303 also failed to consume acetic acid in response to the supplementations of both cations at higher concentrations. In aerobic yeast cultures, mitochondria are critical for ethanol, acetic acid, and glycerol metabolism (3). Mitochondria are affected by osmotic stress, and it is plausible that the K⁺-Na⁺ supplementation affected ion and solute exchanges across the mitochondrial membrane and enzymes involved in utilizing respiratory energy sources (47). The metabolic differences between CEN.PK and W303 are interesting for understanding K⁺-Na⁺ interactions and mechanisms in S. cerevisiae.

Finally, an inverse relationship between cell volume and maximum growth rate was observed among yeasts. This relationship was responsive to salt stress for a given strain. The relationship in growth rate and cell volume was likely due to differences in yeasts’ genomic attributes (as noted under “Strains and culture conditions”) (59). Many smaller cells observed in the image analysis were detected due to samples being collected from exponentially growing cultures, where smaller nascent daughter cells are greater in number than larger mother cells (40, 60). The observed nonnormal volume distribution patterns in the image analysis can be attributed to mother and daughter cell dynamics in yeast cultures. In the stationary phase, median cell and vacuole volumes can be expected to increase in yeast cultures. The homogeneity of vacuole volume and its median value depends on the strain’s growth phase and culture environment. Besides being important in cell division, bud formation is also relevant to the cytosolic pH control, as nascent daughter cells lack the plasma membrane proton ATPase (Pma1) (Fig. 5A) (41, 61). W303 stood out for having a very high standard deviation for cells larger than the median under all tested conditions. The variability in W303 vacuole volume contributes to its overall population heterogeneity. The vacuole-to-cell volume ratio increased for W303 and K. marxianus but not for CEN.PK and R. toruloides, with an increase in cell volume. The vacuole-to-cell volume ratio differences indicated vacuole volume’s relevance for controlling cell volume in yeasts (Fig. S2B, S3B, and S4B). Our findings on cell and vacuole volume are pertinent for using yeasts in aging research. Cell volume is considered proportional to age, and with aging, the vacuole-linked autophagy becomes increasingly dysfunctional (62). The vacuolar dysfunction inhibits efficient catabolism of intravacuolar materials, inducing the vacuole’s enlargement. This dysfunction can also affect mitochondrial functions, causing metabolic diseases (62). The reproducibility of life span extension results using yeast as the model organism is a matter of concern (63). Our results suggest careful consideration of the strain background while using yeasts in aging research. Different yeasts show distinct vacuole-to-cell volume ratios that can potentially affect life span.

In conclusion, the present study describes comparative morphological and physiological features of the nonconventional yeast versus S. cerevisiae in responses to potassium and sodium salt stress. As transport and efflux engineering is an emerging area of biotechnology research, our results will be relevant for further investigations concerning cationic transporters and stress tolerance in yeasts (64). The presented open-source neural network-based method for the optical image analysis will be a valuable community resource for cell and vacuole volume quantifications. Further benchmarking of the molecular mechanisms in the nonconventional yeasts versus those in S. cerevisiae will help design diverse MCFs with a broader substrate-product spectrum, leading to efficient bioprocesses.

MATERIALS AND METHODS

Strains and culture conditions.

This study compared Saccharomyces cerevisiae CEN.PK 113-7D (3), S. cerevisiae CJM 567 isogenic to W303 (65), Kluyveromyces marxianus CBS6556 (16), and Rhodotorula toruloides CCT0783 (66). The genome and chromosome information of the strains in the study is as follow: S. cerevisiae strains consisted of 12-Mb genomes distributed over 16 chromosomes, K. marxianus consisted of an 11-Mb genome spread over 8 chromosomes, and R. toruloides had a 20-Mb genome over 16 chromosomes (9, 10, 53, 67). All experiments were performed in a previously described minimal mineral medium containing 10 g of glucose, 5 g of (NH₄)₂SO₄, 3 g of KH₂PO₄, and 0.5 g of MgSO₄·7H₂O per liter, in addition to 1 ml of trace elements solution and 1 ml of vitamin solution (68). The trace element solution contained, per liter (pH 4), EDTA (sodium salt), 15.0 g; ZnSO₄·7H₂O, 4.5 g; MnCl₂·2H₂O, 0.84 g; CoCl₂·6H₂O, 0.3 g; CuSO₄·5H₂O, 0.3 g; Na₂MoO₄·2H₂O, 0.4 g; CaCl₂·2H₂O, 4.5 g; FeSO₄·7H₂O, 3.0 g; H₃BO₃, 1.0 g; and KI, 0.10 g. The vitamin solution contained, per liter (pH 6.5), biotin, 0.05 g; p-aminobenzoic acid, 0.2 g; nicotinic acid, 1 g; Ca-pantothenate, 1 g; pyridoxine-HCl, 1.0 g; thiamine-HCl, 1.0 g; and myoinositol, 25 g (68). The medium pH was adjusted to 6, with 1.9 ml per liter of 2.0 M KOH. KOH was used because potassium ions were already present at a high enough concentration, and this addition did not change the overall concentration significantly. The final K⁺ concentration in the minimal medium was 25 mM. KCl and NaCl were used to achieve the required K⁺ and Na⁺ concentrations in the supplemented medium. The physiology characterization experiments used 50-ml flasks with vented caps and contained 20 ml of culture volume. If necessary, for downstream processing, cells were washed with 0.9% saline solution. The culture stocks were stored at −80°C in a mixture of 50% YPD (1% yeast extract, 1% peptone, 2% glucose) and 50% glycerol. For all the experiments, cells were precultured in YPD overnight, washed, and then grown in a minimal medium with 1% glucose for 6 h. Afterward, cells were rewashed and inoculated in a required medium to obtain an initial 0.1 optical density (OD) value. Cultivation temperature was maintained at 30°C, and cultivation was conducted under the ambient aerobic condition in a shaker at 200 rpm.

Cell density.

A Hitachi U-1800 spectrophotometer (Japan) and Sarstedt polystyrene cuvettes were used for cell density measurements. The culture aliquots were diluted to keep the optical density at 600 nm (OD₆₀₀) in the range of 0.05 to 0.3, and distilled water was used for the reference.

Metabolite measurements.

For each data point of interest, a 0.25-ml culture volume was collected. In preparation for high-performance liquid chromatography (HPLC), samples were centrifuged twice at 11,000 × g for 5 min and each time transferred to a new tube to remove cells. Afterward, samples were diluted 10 times to decrease the salt concentration, and 60 μl of this diluted sample was used for measurements. An Aminex HPX-87H chromatography column (Bio-Rad) was used for elution of sugars and organic acids (temperature of 45°C, flow rate of 0.6 ml/min, and using a mobile phase of 5 mM sulfuric acid) in a refractive index detector-containing HPLC instrument (Prominence-I, LC-2030 C Plus; Shimadzu) (68).

The carotene measurement was performed according to a previously published protocol (16). Samples (2 ml) were collected from stationary-phase cultures of R. toruloides by centrifugation, washed twice in saline, and resuspended in 1.0 ml of acetone. Cells were lysed with acid-washed glass beads (400 to 650 μm) in a FastPrep homogenizer (3 times, 4 m/s for 20 s) (MP Biomedicals, CA, USA). After centrifugation at 15,000 × g for 5 min, the acetone solution containing carotene was collected. Samples were stored at 4°C until further quantification, which was conducted by using an Acquity ultraperformance liquid chromatography (UPLC) instrument (Waters, MA, USA) equipped with a tunable UV (TUV) detector (Waters) and a C₁₈ column (BEH130, 1.7 μm, 2.1 by 100 mm; Waters). A gradient of 80% to 100% acetone was used in the mobile phase with purified water at a 0.2 ml/min flow rate. Carotene peaks were detected at 450 nm, and quantification was done using a beta-carotene standard at the same condition (Alfa Aesar, MA, USA).

Microplate reader.

Yeasts were precultured as described above. Two hundred microliters of culture medium was used to fill each well in a 96-well plate. The inoculum provided a starting OD₆₀₀ of 0.1. The cultivation was performed using a BioTek Synergy MX microplate reader (BioTek, USA) equipped with a shaker, and the temperature was set to 30°C. The plate reader was controlled by Gen5 ver. 2.04 software, and the culture plate were scanned every 30 min after 15-s high-speed shaking at an absorbance of 600 nm. The absorbance data were imported to a spreadsheet for further analysis.

Growth rate.

The optical cell density data obtained from independent triplicate (or more) experiments were used for calculating the growth rate. In the regression analysis, an exponential curve was fit to a region indicating an exponential growth of the culture during the cultivation. A measurement was considered reproducible when an R² of at least 0.95 was present, and the curve’s slope was taken as the specific growth rate (h⁻¹).

Cell and vacuole volume quantification by image analysis.

Yeasts were precultured as described above, and samples for optical microscopy imaging were collected during the mid-exponential phase, as determined by OD measurements. One milliliter of culture was centrifuged at 3,000 × g for 1 min to obtain a cell pellet. It was resuspended in 3 μl of the same growth medium and transferred to a glass slide for microscopy. A Nikon Eclipse Ci-L (Nikon, Japan) microscope with the Ph3 setting was used for image acquisition. All images were acquired by using Samsung Galaxy S7 Edge (Samsung, South Korea). For image analysis, a custom neural network-based analysis software pipeline was developed in MATLAB and used for segmenting cells in images. The quantification of cell and vacuole volume (femtoliters) was performed by constructing a three-dimensional (3D) model based on a two-dimensional (2D) mask and by optimizing the third dimension for circularity. The median value was used as a representative measure of volume. Both cell and vacuole volume data followed nonnormal distributions. The standard deviations were calculated and represented as distribution bars, indicating heterogeneity in the population. The standard deviations were calculated independently for each half of volume distributions that were spliced into two halves according to respective medians. For the representative volume distribution visualization, 50% standard deviations, in both directions, from the median volume were plotted. The vacuole-to-cell volume ratio was calculated by dividing the median of vacuole volume in each bin. If a bin had fewer than 30 cells, its width was increased until it had 30 cells. The significance analysis of data was performed using the Kruskal-Wallis test, which applies to the nonnormal distribution scenarios, and was implemented in MATLAB.

Cationic transporters analysis.

NCBI nucleotide and protein BLAST were used to determine homology between genes and proteins (69). Genome resources were used for obtaining DNA and protein sequences for comparative analysis: S. cerevisiae CEN.PK 113-7D (9), S. cerevisiae W303 (43), K. marxianus (12), and R. toruloides (44).

Data availability.

The detailed open-source code used for image analysis is available via GitHub (https://github.com/a-ill/Cell-Image-Analysis-Pipeline) and archived in Zenodo (https://doi.org/10.5281/zenodo.4550678).