Physiological and genetic regulation of anhydrobiosis in yeast cells.

Anhydrobiosis is a state of living organisms during which their metabolism is reversibly delayed or suspended due to a high degree of dehydration. Yeast cells, which are widely used in the food industry, may be induced into this state. The degree of viability of yeast cells undergoing the drying process also depends on rehydration. In an attempt to explain the essence of the state of anhydrobiosis and clarify the mechanisms responsible for its course, scientists have described various cellular compounds and structures that are responsible for it. The structures discussed in this work include the cell wall and plasma membrane, vacuoles, mitochondria, and lysosomes, among others, while the most important compounds include trehalose, glycogen, glutathione, and lipid droplets. Various proteins (Stf2p; Sip18p; Hsp12p and Hsp70p) and genes (STF2; Nsip18; TRX2; TPS1 and TPS2) are also responsible for the process of anhydrobiosis. Each factor has a specific function and is irreplaceable, detailed information is presented in this overview.

New concept for conversion of lignocellulose to ethanol and furfural.

Studies on the chemical mechanisms of furfural formation showed the possibility to apply a new differential catalysis of hemicellulose - its depolymerisation and pentose dehydration to furfural. This change led to the increase in furfural yield and essential decrease of cellulose destruction. The lignocellulose residue that remains after the production of furfural may be subjected to enzymatic hydrolysis to glucose and the subsequent fermentation to ethanol. The remaining lignin appeared to be suitable for the production of additional various value-added products including medicinal mushrooms and laccase-containing enzyme complexes. Based on these developments, an innovative concept is proposed for the waste-free use of lignocellulose to obtain various valuable products. KEY POINTS: • New chemical mechanism of furfural production. • New lignocellulose pretreatment does not damage cellulose and lignin. • Lignocellulose may be processed using waste-free technology.

Astrobiology of life on Earth.

Astrobiology is mistakenly regarded by some as a field confined to studies of life beyond Earth. Here, we consider life on Earth through an astrobiological lens. Whereas classical studies of microbiology historically focused on various anthropocentric sub-fields (such as fermented foods or commensals and pathogens of crop plants, livestock and humans), addressing key biological questions via astrobiological approaches can further our understanding of all life on Earth. We highlight potential implications of this approach through the articles in this Environmental Microbiology special issue 'Ecophysiology of Extremophiles'. They report on the microbiology of places/processes including low-temperature environments and chemically diverse saline- and hypersaline habitats; aspects of sulphur metabolism in hypersaline lakes, dysoxic marine waters, and thermal acidic springs; biology of extremophile viruses; the survival of terrestrial extremophiles on the surface of Mars; biological soils crusts and rock-associated microbes of deserts; subsurface and deep biosphere, including a salticle formed within Triassic halite; and interactions of microbes with igneous and sedimentary rocks. These studies, some of which we highlight here, contribute to our understanding of the spatiotemporal reach of Earth'sfunctional biosphere, and the tenacity of terrestrial life. Their findings will help set the stage for future work focused on the constraints for life, and how organisms adapt and evolve to circumvent these constraints.

A Crucial Role of Mitochondrial Dynamics in Dehydration Resistance in Saccharomyces cerevisiae.

Mitochondria are dynamic organelles as they continuously undergo fission and fusion. These dynamic processes conduct not only mitochondrial network morphology but also activity regulation and quality control. Saccharomyces cerevisiae has a remarkable capacity to resist stress from dehydration/rehydration. Although mitochondria are noted for their role in desiccation tolerance, the mechanisms underlying these processes remains obscure. Here, we report that yeast cells that went through stationary growth phase have a better survival rate after dehydration/rehydration. Dynamic defective yeast cells with reduced mitochondrial genome cannot maintain the mitochondrial activity and survival rate of wild type cells. Our results demonstrate that yeast cells balance mitochondrial fusion and fission according to growth conditions, and the ability to adjust dynamic behavior aids the dehydration resistance by preserving mitochondria.

Anhydrobiosis in yeast: role of cortical endoplasmic reticulum protein Ist2 in Saccharomyces cerevisiae cells during dehydration and subsequent rehydration.

Two Saccharomyces cerevisiae strains, BY4741 and BY4741-derived strain lacking the IST2 gene (ist2Δ), were used to characterise the possible role of cortical endoplasmic reticulum (ER) protein Ist2 upon cell dehydration and subsequent rehydration. For the first time, we show that not only protein components of the plasma membrane (PM), but also at least one ER membrane protein (Ist2) play an important role in the maintenance of the viability of yeast cells during dehydration and subsequent rehydration. The low viability of the mutant strain ist2∆ upon dehydration-rehydration stress was related to the lack of Ist2 protein in the ER. We revealed that the PM of ist2∆ strain is not able to completely restore its molecular organisation during reactivation from the state of anhydrobiosis. As the result, the permeability of the PM remains high regardless of the type of reactivation (rapid or gradual rehydration). We conclude that ER protein Ist2 plays an important role in ensuring the stability of molecular organisation and functionality of the PM during dehydration-rehydration stress. These results indicate an important role of ER-PM interactions during cells transition into the state of anhydrobiosis and the subsequent restoration of their physiological activities.

Changes in Energy Status of Saccharomyces cerevisiae Cells during Dehydration and Rehydration.

Anhydrobiosis is the state of life when cells are exposed to waterless conditions and gradually cease their metabolism. In this study, we determined the sequence of events in Saccharomyces cerevisiae energy metabolism during processes of dehydration and rehydration. The intensities of respiration and acidification of the medium, the amounts of phenyldicarbaundecaborane (PCB-) bound to yeast membranes, and the capabilities of cells to accumulate K+ were assayed using an electrochemical monitoring system, and the intracellular content of ATP was measured using a bioluminescence assay. Mesophilic, semi-resistant to desiccation S. cerevisiae strain 14 and thermotolerant, very resistant to desiccation S. cerevisiae strain 77 cells were compared. After 22 h of drying, it was possible to restore the respiration activity of very resistant to desiccation strain 77 cells, especially when glucose was available. PCB- binding also indicated considerably higher metabolic activity of dehydrated S. cerevisiae strain 77 cells. Electrochemical K+ content and medium acidification assays indicated that permeabilization of the plasma membrane in cells of both strains started almost simultaneously, after 8-10 h of desiccation, but semi-resistant strain 14 cells maintained the K+ gradient for longer and more strongly acidified the medium. For both cells, the fast rehydration in water was less efficient compared to reactivation in the growth medium, indicating the need for nutrients for the recovery. Higher viability of strain 77 cells after rehydration could be due to the higher stability of their mitochondria.

Carotenoids and Some Other Pigments from Fungi and Yeasts.

Carotenoids are an essential group of compounds that may be obtained by microbiological synthesis. They are instrumental in various areas of industry, medicine, agriculture, and ecology. The increase of carotenoids' demand at the global market is now essential. At the moment, the production of natural carotenoids is more expensive than obtaining their synthetic forms, but several new approaches/directions on how to decrease this difference were developed during the last decades. This review briefly describes the information accumulated until now about the beneficial effects of carotenoids on human health protection, their possible application in the treatments of various diseases, and their use in the food and feed industry. This review also describes some issues that are linked with biotechnological production of fungal and yeasts carotenoids, as well as new approaches/directions to make their biotechnological production more efficient.

Correlation of Statistical Distributions of the Dimension of Yeast Cells Attached to the Substrate and Its Surface Electrical Potential.

The ability of cells to adhere to substrates is an important factor for the effectiveness of biotechnologies and bioimplants. This research demonstrates that the statistical distribution of the sizes of the cells (Saccharomyces cerevisiae) attached to the substrate surface correlates with the statistical distribution of electrical potential on the substrate's surface. Hypothetically, this behavior should be taken into consideration during the processing of surfaces when cell adhesion based on cell size is required.

Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast.

BACKGROUND: Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. RESULTS: In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae, were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha. CONCLUSIONS: The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulting strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

Microbial lag phase can be indicative of, or independent from, cellular stress.

Measures of microbial growth, used as indicators of cellular stress, are sometimes quantified at a single time-point. In reality, these measurements are compound representations of length of lag, exponential growth-rate, and other factors. Here, we investigate whether length of lag phase can act as a proxy for stress, using  a number of model systems (Aspergillus penicillioides; Bacillus subtilis; Escherichia coli; Eurotium amstelodami, E. echinulatum, E. halophilicum, and E. repens; Mrakia frigida; Saccharomyces cerevisiae; Xerochrysium xerophilum; Xeromyces bisporus) exposed to mechanistically distinct types of cellular stress including low water activity, other solute-induced stresses, and dehydration-rehydration cycles. Lag phase was neither proportional to germination rate for X. bisporus (FRR3443) in glycerol-supplemented media (r2 = 0.012), nor to exponential growth-rates for other microbes. In some cases, growth-rates varied greatly with stressor concentration even when lag remained constant. By contrast, there were strong correlations for B. subtilis in media supplemented with polyethylene-glycol 6000 or 600 (r2 = 0.925 and 0.961), and for other microbial species. We also  analysed data from independent studies of food-spoilage fungi under glycerol stress (Aspergillus aculeatinus and A. sclerotiicarbonarius); mesophilic/psychrotolerant bacteria under diverse, solute-induced stresses (Brochothrix thermosphacta, Enterococcus faecalis, Pseudomonas fluorescens, Salmonella typhimurium, Staphylococcus aureus); and fungal enzymes under acid-stress (Terfezia claveryi lipoxygenase and Agaricus bisporus tyrosinase). These datasets also exhibited diversity, with some strong- and moderate correlations between length of lag and exponential growth-rates; and sometimes none. In conclusion, lag phase is not  a reliable measure of stress because length of lag and growth-rate inhibition are sometimes highly correlated, and sometimes not at all.

Anhydrobiosis in yeasts: Glutathione synthesis by yeast Ogataea (Hansenula) polymorpha cells after their dehydration-rehydration.

The possibility of using active dry microbial preparations in biotechnological processes is essential for the development of new modern industrial technologies. In this study, we show the possibility of obtaining such preparations of the genetically engineered yeast strain Ogataea (Hansenula) polymorpha with glutathione overproduction. Special pre-treatment involving the gradual rehydration of dry cells in water vapour led to the restoration/reactivation of almost 100% of dehydrated cells. Furthermore, dry cells do not lose their viability during storage at room temperatures. Application of dry cells as the inoculum provides the same levels of glutathione synthesis as that of a native yeast culture.

Anhydrobiosis in yeasts: Psychrotolerant yeasts are highly resistant to dehydration.

Yeast cells are able to transition into a state of anhydrobiosis (temporary reversible suspension of metabolism) under conditions of desiccation. One of the most efficient approaches for understanding the mechanisms underlying resistance to dehydration-rehydration is to identify yeasts, which are stable under such treatments, and compare them with moderately resistant species and strains. In the current study, we investigated the resistance to dehydration-rehydration of six psychrotolerant yeast strains belonging to two species. All studied strains of Solicoccozyma terricola and Naganishia albida were found to be highly resistant to dehydration-rehydration. The viability of S. terricola strains was close to 100%. Such results have not been previously reported in studies of anhydrobiosis in yeasts. The plasma membrane changes, revealed by determining its permeability under various rehydration conditions, were also surprisingly minimal. Thus, the high level of resistance of psychrotolerant yeast strains might be related to the chemical composition and molecular organisation of their plasma membranes. Aside from plasma membrane characteristics, other important factors may also influence the maintenance of yeast cell viability under conditions of dehydration-rehydration.

Anhydrobiosis: Inside yeast cells.

Under natural conditions yeast cells as well as other microorganisms are regularly subjected to the influence of severe drought, which leads to their serious dehydration. The dry seasons are then changed by rains and there is a restoration of normal water potential inside the cells. To survive such seasonal changes a lot of vegetative microbial cells, which belong to various genera and species, may be able to enter into a state of anhydrobiosis, in which their metabolism is temporarily and reversibly suspended or delayed. This evolutionarily developed adaptation to extreme conditions of the environment is widely used for practical goals - for conservation of microorganisms in collections, for maintenance and long storage of different important strain-producers and for other various biotechnological purposes. This current review presents the most important data obtained mainly in the studies of the structural and functional changes in yeast cells during dehydration. It describes the changes of the main organelles of eukaryotic cells and their role in cell survival in a dry state. The review provides information regarding the role of water in the structure and functions of biological macromolecules and membranes. Some important intracellular protective reactions of eukaryotic organisms, which were revealed in these studies and may have more general importance, are also discussed. The results of the studies of yeast anhydrobiosis summarized in the review show the possibilities of improving the conservation and long-term storage of various microorganisms and of increasing the quality of industrially produced dry microbial preparations.

Bioethanol and lipid production from the enzymatic hydrolysate of wheat straw after furfural extraction.

This study investigates biofuel production from wheat straw hydrolysate, from which furfural was extracted using a patented method developed at the Latvian State Institute of Wood Chemistry. The solid remainder after furfural extraction, corresponding to 67.6% of the wheat straw dry matter, contained 69.9% cellulose of which 4% was decomposed during the furfural extraction and 26.3% lignin. Enzymatic hydrolysis released 44% of the glucose monomers in the cellulose. The resulting hydrolysate contained mainly glucose and very little amount of acetic acid. Xylose was not detectable. Consequently, the undiluted hydrolysate did not inhibit growth of yeast strains belonging to Saccharomyces cerevisiae, Lipomyces starkeyi, and Rhodotorula babjevae. In the fermentations, average final ethanol concentrations of 23.85 g/l were obtained, corresponding to a yield of 0.53 g ethanol per g released glucose. L. starkeyi generated lipids with a rate of 0.08 g/h and a yield of 0.09 g per g consumed glucose. R. babjevae produced lipids with a rate of 0.18 g/h and a yield of 0.17 per g consumed glucose. In both yeasts, desaturation increased during cultivation. Remarkably, the R. babjevae strain used in this study produced considerable amounts of heptadecenoic, α,- and γ-linolenic acid.

Activity of the α-glucoside transporter Agt1 in Saccharomyces cerevisiae cells during dehydration-rehydration events.

Microbial cells can enter a state of anhydrobiosis under desiccating conditions. One of the main determinants of viability during dehydration-rehydration cycles is structural integrity of the plasma membrane. Whereas much is known about phase transitions of the lipid bilayer, there is a paucity of information on changes in activity of plasma membrane proteins during dehydration-rehydration events. We selected the α-glucoside transporter Agt1 to gain insights into stress mechanisms/responses and ecophysiology during anhydrobiosis. As intracellular water content of S. cerevisiae strain 14 (a strain with moderate tolerance to dehydration-rehydration) was reduced to 1.5 g water/g dry weight, the activity of the Agt1 transporter decreased by 10-15 %. This indicates that functionality of this trans-membrane and relatively hydrophobic protein depends on water. Notably, however, levels of cell viability were retained. Prior incubation in the stress protectant xylitol increased stability of the plasma membrane but not Agt1. Studies were carried out using a comparator yeast which was highly resistant to dehydration-rehydration (S. cerevisiae strain 77). By contrast to S. cerevisiae strain 14, there was no significant reduction of Agt1 activity in S. cerevisiae strain 77 cells. These findings have implications for the ecophysiology of S. cerevisiae strains in natural and industrial systems.

Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd.

Application of anhydrobiosis and dehydration of yeasts for non-conventional biotechnological goals.

Dehydration of yeast cells causes them to enter a state of anhydrobiosis in which their metabolism is temporarily and reversibly suspended. This unique state among organisms is currently used in the production of active dry yeasts, mainly used in baking and winemaking. In recent decades non-conventional applications of yeast dehydration have been proposed for various modern biotechnologies. This mini-review briefly summarises current information on the application of dry yeasts in traditional and innovative fields. It has been shown that dry yeast preparations can be used for the efficient protection, purification and bioremediation of the environment from heavy metals. The high sorption activity of dehydrated yeasts can be used as an interesting tool in winemaking due to their effects on quality and taste. Dry yeasts are also used in agricultural animal feed. Another interesting application of yeast dehydration is as an additional stage in new methods for the stable immobilisation of microorganisms, especially in cases when biotechnologically important strains have no affinity with the carrier. Such immobilisation methods also provide a new approach for the successful conservation of yeast strains that are very sensitive to dehydration. In addition, the application of dehydration procedures opens up new possibilities for the use of yeast as a model system. Separate sections of this review also discuss possible uses of dry yeasts in biocontrol, bioprotection and biotransformations, in analytical methods as well as in some other areas.

Drying enhances immunoactivity of spent brewer's yeast cell wall ß-D-glucans.

Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast ß-D-glucans as BRM, and drying as an efficient pretreatment to increase ß-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified ß-D-glucan. However, drying increased purified ß-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted ß-D-glucan. This is corroborated with FT-IR analyses of the ß-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality ß-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast ß-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran.

The role of glycerol transporters in yeast cells in various physiological and stress conditions.

Small and uncharged glycerol is an important molecule for yeast metabolism and osmoadaptation. Using a series of S. cerevisiae BY4741-derived mutants lacking genes encoding a glycerol exporter (Fps1p) and/or importer (Stl1p) and/or the last kinase of the HOG pathway (Hog1p), we studied their phenotypes and various physiological characteristics with the aim of finding new roles for glycerol transporters. Though the triple mutant hog1Δ stl1Δ fps1Δ was viable, it was highly sensitive to various stresses. Our results showed that the function of both Stl1p and Fps1p transporters contributes to the cell ability to survive during the transfer into the state of anhydrobiosis, and that the deletion of FPS1 decreases the cell's tolerance of hyperosmotic stress. The deletion of STL1 results in a slight increase in cell size and in a substantial increase in intracellular pH. Taken together, our results suggest that the fluxes of glycerol in both directions across the plasma membrane exist in yeast cells simultaneously, and the export or import predominates according to the actual specific conditions.

Survival kit of Saccharomyces cerevisiae for anhydrobiosis.

Yeast cells are well adapted to interfacial habitats, such as the surfaces of soil or plants, where they can resist frequent fluctuations between wet and dry conditions. Saccharomyces cerevisiae is recognized as an anhydrobiotic organism, and it has been the subject of numerous studies that aimed to elucidate this ability. Extensive data have been obtained from these studies based on a wide range of experimental approaches, which have added significantly to our understanding of the cellular bases and mechanisms of resistance to desiccation. The aim of this review is to provide an integrated view of these mechanisms in yeast and to describe the survival kit of S. cerevisiae for anhydrobiosis. This kit comprises constitutive and inducible mechanisms that prevent cell damage during dehydration and rehydration. This review also aims to characterize clearly the phenomenon of anhydrobiosis itself based on detailed descriptions of the causes and effects of the constraints imposed on cells by desiccation. These constraints mainly lead to mechanical, structural, and oxidative damage to cell components. Considerations of these constraints and the possible utilization of components of the survival kit could help to improve the survival of sensitive cells of interest during desiccation.