Introduction

One of the most prominent discoveries of Antonie van Leeuwenhoek was the state of anabiosis (cryptobiosis), which was made on 1 September 1701. In spite of the long history of studies of this unique natural phenomenon, many issues linked to mechanisms of anabiosis have still not been elucidated. Yeast cells are living organisms that can transit into a state of anabiosis and more precisely into a state of anhydrobiosis in unfavourable environmental conditions, which is linked to a temporary reversible delay of metabolism as the result of cell desiccation (Beker and Rapoport 1987). Even though this phenomenon is widely used in biotechnology for the production of different dry biopreparations, including active bakers’ dry yeast, the intracellular mechanisms that allow for a high viability in the dry state have still not been completely elucidated. Currently, it is known that all intracellular organelles are subjected to dehydration processes that induce more or less serious structural and functional changes (Beker and Rapoport 1987). Special attention in studies of yeast anhydrobiosis has been devoted to the state of intracellular membranes (mainly the plasma membrane) and to intracellular protective reactions (Beker and Rapoport 1987; Guzhova et al. 2008). Strong invaginations of the plasma membrane have been found in dehydrated cells (Rapoport and Kostrikina 1973; Biryusova and Rapoport 1978), together with significant increases in membrane permeability (Beker et al. 1984; Novichkova and Rapoport 1984; Rapoport et al. 1995, 1997). The conclusion was reached that the nature of membrane sterols governs the mechanical changes in yeast plasma membranes during dehydration stress. The accumulation of ergosterol might be necessary for the resistance of yeast cells to drying (Dupont et al. 2011). Some protective compounds (trehalose, polyols) are synthesised by cells of yeast that belongs to various genera, to promote the maintenance of the molecular organisation of membranes during cell dehydration–rehydration and lead to a decrease in plasma membrane permeability (Crowe et al. 1984; Rapoport et al. 1988, 2009). These results were obtained from studies of bakers’ yeast grown in aerobic conditions; there have been no investigations to date of the influence of dehydration on Saccharomyces cerevisiae cells grown in anaerobic conditions. As a result, commercial preparations of aerobically grown bakers’ active dry yeasts have a very high quality, but the desiccation of S. cerevisiae grown in anaerobic conditions still presents serious problems, and this yeast has an extremely low viability in the dehydrated state. However, yeasts grown under anaerobic (oxygen-limited) conditions are used for production of beer, wine and ethanol/bioethanol and dry active anaerobic yeasts can be used in the same processes.

It is well known that the physiology of S. cerevisiae cells grown in aerobic or anaerobic conditions differs significantly. Molecular oxygen is not only essential for respiration, but is also required in several biosynthetic pathways, such as those for haem, sterols, unsaturated fatty acids, pyrimidines and deoxyribonucleotides (Snoek and Steensma 2007; Bruckmann et al. 2009). Transcriptional analyses have shown that the expression levels of ~500 genes differ significantly when aerobic and anaerobic cultures are compared (Ter Linde et al. 1999; Kwast et al. 2002; Piper et al. 2002; Tai et al. 2005; Bruckmann et al. 2009). It was also that levels of most of the 21 quantified glycolytic proteins were twofold to tenfold higher in anaerobic than aerobic cultures (de Groot et al. 2007; Bruckmann et al. 2009). The lipid composition of the membrane under anaerobic conditions is different from that of cells grown under aerobic conditions. In anaerobically grown S. cerevisiae, plasma membranes contain more saturated fatty acids, but less total sterol, ergosterol and squalene. These differences can be explained by the inability of the cell to synthesise these compounds without oxygen (Nurminen et al. 1975; Snoek and Steensma 2007). The yeast S. cerevisiae is auxotrophic for ergosterol in the absence of oxygen. It was shown that complex changes in the esterification of exogenously supplied sterols were also induced by anaerobiosis. The utilisation of oleic acid for sterol esterification was significantly impaired in anaerobic cells. These results indicate that sterol esters might fulfil different roles in aerobic and anaerobic cells (Valachovi et al. 2001). The aim of the present investigation was to determine whether it is possible to induce S. cerevisiae cells grown in conditions with severe oxygen limitation to enter a state of anhydrobiosis.

Materials and methods

The yeast strain S. cerevisiae 14 from the collection of the Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia was used in this study. Yeast cells were cultivated in 500-mL flasks, which contained 450 mL nutrient medium at 30 °C, without shaking. Under these conditions, the oxygen supply to cells is very poor. We defined the applied cultivation mode as being anaerobic conditions or severe oxygen limitation and use these two terms in this work synonymously. To reveal the resistance of S. cerevisiae cultures grown in anaerobic conditions to dehydration, they were cultivated in two different nutrient media. The first nutrient medium contained (in g L−1): MgSO4 0.7; NaCl 0.5; (NH4)2SO4 3.7; KH2PO4 1.0; K2HPO4 0.13; molasses 42. The pH of the nutrient medium was adjusted to pH 5.0 using H2SO4. The second nutrient medium contained unhopped beer wort (4° Balling). Yeast cells were collected when they reached the stationary phase. A total of five experiments were conducted with each nutrient medium.

The cells were centrifuged (2,600×g, 10 min) and separated from the culture liquid. Bakers’ yeast biomass was compressed and suspended in different solutions and incubated at 30 °C in flasks on an orbital shaker for 3 h. Control samples were incubated in sterile water in the same conditions as the other samples. The hyperosmotic solutions were prepared by addition of sugar (lactose) or sugar alcohols (xylitol or glycerol), were provided at different molarities (0.25, 0.5, 0.75, and 1 M). Yeast cells were not washed after these treatments. Other solutions contained 100 mM antioxidant—glutathione, or 100 mM sugar—trehalose. Experiments were also performed by adding 100 µM or 100 mM glutathione to the nutrient medium before the start of yeast cultivation.

Bakers’ yeast biomass was compressed, extruded through a sieve, and subjected to convective dehydration in an oven at 30 °C, until the cell’s water content decreased to 8–10 %. The dehydration rate was changed by regulating the conductivity of air access to the yeast samples. Residual moisture was determined by drying to constant weight at 105 °C. At residual moistures of 8–10 %, yeast cells are presumed to be in a state of anhydrobiosis if they still maintain viability at such residual moistures (Beker and Rapoport 1987). Survival rates of the dehydrated yeast cells were determined by fluorescence microscopy with the fluorochrome primulin (Rapoport and Meissel 1985). Different types of dry yeast rehydration (fast and slow) were performed, usually within 1 week following yeast dehydration. During this storage period, samples of dehydrated yeast cells were kept at room temperature. Slow rehydration of dry yeasts was performed by maintaining yeast for 2 h at 37 °C in water vapour. Rapid rehydration of dry samples was performed by keeping S. cerevisiae cells in distilled water for 10 min at room temperature.

A short scheme of experiments performed in this study is shown in Fig. 1. All experiments were performed at least five times, and mean values ± SD are presented.

Fig. 1
figure 1

The general scheme of experiments performed in this study

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Results and discussion

When two different nutrient media were used, the viability of S. cerevisiae cultures grown in the conditions with severe oxygen limitation after dehydration treatment was within the range of 0–2 %, independent of the nutrient medium. It was not possible to increase these viability rates by slow, gradual rehydration of dry cells with water vapour. It is widely accepted that the rehydration stage is very important for the successful recovery of dehydrated yeasts (Beker and Rapoport 1987; Soubeyrand et al. 2005, 2006; Simonin et al. 2007; Díaz-Hellín et al. 2013). It was previously shown in experiments performed with different yeasts grown in aerobic conditions, that slow gradual rehydration of dry cells, in accordance with the model of changes in the molecular organisation of intracellular membranes (Crowe et al. 1989), leads to the restoration of amounts of bound water that are necessary in intracellular membranes (primarily the plasma membrane) (Rapoport et al. 1995, 1997). As a result, a decrease in the membrane lipid-phase transition temperature was reached. In turn, this was accompanied by a significant decrease in plasma membrane permeability during subsequent rehydration of cells in bulk water and promoted the repair of membrane damage. Nevertheless, in our experiments, such an approach did not generate positive results. Therefore, if membrane damage is also one reason for cell death in anaerobic conditions, it is reasonable to suppose that damage upon dehydration is so serious that it becomes irreversible.

In experiments with aerobically grown yeasts, a slower dehydration usually led to a higher viability of dry cells. This effect was attributed to the activation of intracellular protective reactions as well as to a decrease in intracellular membrane damage (Beker and Rapoport 1987; Dupont et al. 2010). Therefore, we attempted to increase the viability of dry cells by changing the rate of dehydration. However, we did not observe a higher viability of dry anaerobically grown cells, which still remained at about 2 %.

Our previous experiments performed with aerobically grown yeast, showed that the resistance to dehydration of a non-resistant population can be successfully increased by incubating the cells in solutions of sucrose or lactose with a higher osmotic pressure (Rapoport and Beker 1983). In these conditions, sugar alcohols (mannitol, inositol and others) are synthesised in the cells and the phase-transition temperature of membrane lipids decreases (Rapoport et al. 1988, 2009). We attempted to use the same approach in this study for anaerobically grown yeast cultures. With this goal, anaerobically grown S. cerevisiae cells were incubated (before dehydration) in 0.25, 0.5, 0.75 or 1 M lactose for 3 h. In these experiments, a small increase in the resistance of the cells to dehydration was reached following incubation with 1 M lactose. After dehydration of these cultures, their viability was already about 10 %. Gradual rehydration of the same dehydrated yeast cultures in water vapour did not increase the proportion of viable cells. Even though the increase in viability in these conditions was rather small, we interpret these results as showing the possibility to influence the state of the cells by incubation in solutions with a high osmotic pressure. Therefore, we supplied the cells artificially with protective compounds that belong to different groups; antioxidants (glutathione) and compounds that can substitute for water hydroxyl groups (trehalose, xylitol and glycerol).

When glutathione was added to the nutrient medium prior to S. cerevisiae cultivation or was used for incubation in solutions of yeast biomass, no increase in cell viability was observed. As glutathione maintains the intracellular redox balance during the dehydration of aerobically grown yeast (Espindola et al. 2003), presumably oxidative stress is not the main factor responsible for irreversible cell damage at dehydration of yeast cultures grown in conditions with severe oxygen limitation.

To test the influence of trehalose on cell survival, the cells were incubated in 100 mM trehalose. No increase in the viability of the dry cells following rapid rehydration was noted, and this figure did not exceed 7 ± 2 %. When the dry cells were gradually rehydrated, a viability of 30 ± 4 % of cells was reached. This demonstrated that S. cerevisiae grown in anaerobic conditions can be converted into a state of anhydrobiosis. The hypothesis was proposed that trehalose, whose protective effects are usually explained by preventing the increase in the gel-to-liquid phase transition temperature, Tm, of dehydrated lipid bilayers (Crowe et al. 1984, 1992, 1998), also possesses the same properties for yeast cultures grown in anaerobic conditions. According to the recently modified water replacement hypothesis, trehalose stabilises dry membranes by preventing the decrease in spacing between membrane lipids under dehydration and causes a concentration-dependent increase in the area per lipid (APL), accompanied by an increase in the fluidity of the bilayer core (Crowe et al. 1984, 1992; Golovina et al. 2009, 2010). Possibly, this trehalose effect also occurs for yeast grown anaerobically. In conditions of severe oxygen limitation, this protective effect occurs mainly following the slow rehydration of dry cells. We suggest that in this case, changes in and/or damage to membranes and macromolecules that arise during cell dehydration are essentially more serious and can only be repaired following the return of the cell to a sufficient amount of bound water.

If the protective effect of trehalose can be really explained via the water replacement hypothesis, further experiments with xylitol and glycerol should also lead to a similar increase in resistance to dehydration in anaerobically grown yeast cells. The experiments showed that the best results for yeast grown in severe oxygen-limiting conditions were obtained when 1 M xylitol or 1 M glycerol were added to the incubation solution. Indeed, a concentration-dependent increase in cell viability was observed when cells were preincubated with these sugar alcohols (Figs. 2, 3). Cells pretreated with 1 M xylitol showed survival rates of 34 % when they were rapidly rehydrated and 52 % following slow rehydration (Fig. 2). Cells that were pretreated with 1 M glycerol and were slowly rehydrated had a survival rate of 60 % (Fig. 3). Therefore, these experiments confirmed that anhydrobiosis can also be reached for S. cerevisiae cells grown under strong oxygen limitation, but only if they are pre-treated before dehydration. The results presented in this study support the revised water replacement hypothesis. However, there are essential differences in the effects of trehalose and of xylitol and glycerol. This might be explained by the hypothesis that both xylitol and glycerol possess double or multiple effects. In addition to the effects of trehalose in the case of xylitol and glycerol, additional effects linked with incubating the cells in hyperosmotic solutions exist. As we showed previously (Rapoport and Beker 1983; Rapoport et al. 1988, 2009), we presume that in these conditions, the complex of intracellular protective reactions is switched on. Our results indicated that there are different modes of action for glycerol and xylitol. Glycerol formation is vital for the reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in S. cerevisiae (Modig et al. 2007). The accumulated glycerol functions as an osmolyte, preventing loss of turgor pressure of the cell (Blomberg and Adler 1992; Modig et al. 2007). Therefore, we can assume that the amount of glycerol in anaerobically grown cells in our conditions is not sufficient for the maintenance of all its functions and its additional accumulation during the incubation procedure is necessary. Our results showed that the effect of glycerol was only evident after a preliminary gradual rehydration of cells. Probably, this leads to a decrease in the loss of glycerol from cells during the rehydration stage.

Fig. 2
figure 2

The viability of yeast cells grown anaerobically and subjected to incubation in various solutions of xylitol (0.25, 0.5, 0.75, and 1 M) before drying and subjected to rapid or slow rehydration

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Fig. 3
figure 3

The viability of yeast cells grown anaerobically and subjected to incubation in various solutions of glycerol (0.25, 0.5, 0.75, and 1 M) before drying and subsequent rapid or slow rehydration

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As already mentioned, yeast grown in aerobic and anaerobic conditions differ significantly. It is very important that the plasma membranes of cells cultivated in anaerobic conditions contain more saturated fatty acids, less total sterol, less ergosterol and less squalene (Nurminen et al. 1975; Shobayashi et al. 2007; Snoek and Steensma 2007). It was shown that ergosterol is extremely important for the maintenance of cell resistance to different stresses including dehydration (Dupont et al. 2011, 2012). Ergosterol can act as a mechanical reinforcer in membranes and also as an antioxidant that protects lipids (Dupont et al. 2011, 2012). Therefore, further studies should certainly be linked with attempts to monitor possible changes and modifications of the membrane lipid profile in conditions that lead to an increase in cell resistance to dehydration and which were found in this study. One important question is whether the treatments we have employed, which essentially increased the resistance of anaerobically-grown cells to dehydration–rehydration, changed the composition of membranes to reflect those of aerobically grown S. cerevisiae cells. Together, our results show that cells of S. cerevisiae grown in anaerobic (or oxygen-limiting) conditions can be induced to enter the state of anhydrobiosis. We assume that the integrity of cell membranes in anaerobically grown yeast is the main factor causing cell resistance to dehydration–rehydration. These issues are currently being studied in our laboratory.