Preparation of selenium yeasts I. Preparation of selenium-enriched Saccharomyces cerevisiae

Selenium (Se) is an essential micronutrient for human and animal organisms. Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable.Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast.Using a culture medium supplemented with 30 μg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200–1400 μg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method. In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast. As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content. The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast. A 0.40–0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality. Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5–6% at the expense, however, of approximately a 20% decrease in the final biomass.     

Preparation of selenium yeasts I. Preparation of selenium-enriched Saccharomyces cerevisiae.

Selenium (Se) is an essential micronutrient for human and animal organisms. Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable. Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast. Using a culture medium supplemented with 30 microg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200-1400 microg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method. In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast. As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content. The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast. A 0.40-0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality. Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5-6% at the expense, however, of approximately a 20% decrease in the final biomass.     

Accumulation and metabolism of selenium by yeast cells

This paper examines the process of selenium bioaccumulation and selenium metabolism in yeast cells. Yeast cells can bind elements in ionic from the environment and permanently integrate them into their cellular structure. Up to now, Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica yeasts have been used primarily in biotechnological studies to evaluate binding of minerals. Yeast cells are able to bind selenium in the form of both organic and inorganic compounds. The process of bioaccumulation of selenium by microorganisms occurs through two mechanisms: extracellular binding by ligands of membrane assembly and intracellular accumulation associated with the transport of ions across the cytoplasmic membrane into the cell interior. During intracellular metabolism of selenium, oxidation, reduction, methylation, and selenoprotein synthesis processes are involved, as exemplified by detoxification processes that allow yeasts to survive under culture conditions involving the elevated selenium concentrations which were observed. Selenium yeasts represent probably the best absorbed form of this element. In turn, in terms of wide application, the inclusion of yeast with accumulated selenium may aid in lessening selenium deficiency in a diet.     

Selenium tolerance of yeasts

Selenium tolerance of yeasts widely varies: the growth of some yeasts can be inhibited by a selenium concentration as low as 10(-4) M, whereas others can grow in the presence of 10(-1) M selenium. Homogeneous yeast taxa are characterized by a certain level of selenium tolerance, and heterogeneous taxa show a variable level of tolerance to selenium. In general, ascomycetous yeasts are more tolerant to selenium than basidiomycetous yeasts. Among the ascomycetous yeasts, the genera Dekkera and Schizosaccharomyces exhibited the lowest and the species Candida maltosa, Hanseniaspora valbyensis, Kluyveromyces marxianus, and Yarrowia lipolytica the highest tolerance to selenium. Among the basidiomycetous yeasts, the genera Bullera, Cryptococcus, and Holtermannia showed the lowest and the species Cryptococcus curvatus, Cr. humicola, and Trichosporon spp. the highest tolerance to selenium. The selenium tolerance of yeasts depends on the composition of the growth medium, in particular, on the presence of sulfate, sulfur-containing amino acids, and glutamine in the medium.     

Selenium Tolerance of Yeasts

Selenium tolerance of yeasts widely varies: the growth of some yeasts can be inhibited by a selenium concentration as low as 10^–4 M, whereas others can grow in the presence of 10^–1 M selenium. Homogeneous yeast taxa are characterized by a certain level of selenium tolerance, and heterogeneous taxa show a variable level of tolerance to selenium. In general, ascomycetous yeasts are more tolerant to selenium than basidiomycetous yeasts. Among the ascomycetous yeasts, the genera Dekkera and Schizosaccharomyces exhibited the lowest and the species Candida maltosa, Hanseniaspora valbyensis, Kluyveromyces marxianus, and Yarrowia lipolytica the highest tolerance to selenium. Among the basidiomycetous yeasts, the genera Bullera, Cryptococcusand Holtermannia showed the lowest and the species Cryptococcus curvatus, Cr. humicola, and Trichosporon spp. the highest tolerance to selenium. The selenium tolerance of yeasts depends on the composition of the growth medium, in particular, on the presence of sulfate, sulfur-containing amino acids, and glutamine in the medium.     

Selenium incorporation into Saccharomyces cerevisiae cells: a study of different incorporation methods

AIMS: To study the effects of the selenium enrichment protocols in yeast at various points in the cell cycle, total selenium accumulation and the forms of selenium incorporated. METHODS AND RESULTS: The use of selenized yeast as enriched selenium supplements in human nutrition has become a topic of increasing interest over the last decade. Four enrichment procedures have been evaluated using sodium selenite as the selenium source: enrichment during the growth phase; enrichment at the non-growth phase, both of these at different selenium levels; enrichment by seeding in a fermentable carbon source (glucose); Se-enrichment with a non-fermentable carbon source (glycerol). A nitric acid digestion of the yeast samples prepared under different conditions has been performed in order to evaluate the total selenium incorporated into the yeast cells. Also, an enzymatic digestion of the yeast samples with pepsin has been carried out as an initial step to begin the process of determining which of the different possible selenium species are formed. The cell count evaluations of the selenium-enriched yeast showed that the growth phase, seeding and the use of YEPG media is influenced by the addition of Se, while the non-growth phase is not. Total selenium incorporation studies showed that seeding the yeast permits more accumulation of selenium. Speciation studies of the enriched yeast showed that the growth phase increases the formation of L-Se-methionine. CONCLUSIONS: When the aim of enriching yeast with selenium is the formation of L-Se-methionine, the best enrichment procedure is using the growth phase with small concentrations of sodium selenite. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of selenium supplements is widespread and most of the supplements use selenium-enriched yeast in their formulation. Studies made on supplements do not have the appropriate Se-species for optimal absorption in the human body. This study presents and compares methods for the best selenium yeast enrichment that could ultimately be used in selenium supplement formulations.     

Marine yeasts as biocontrol agents and producers of bio-products

As some species of marine yeasts can colonize intestine of marine animals, they can be used as probiotics. It has been reported that β-glucans from marine yeast cells can be utilized as immuno-stimulants in marine animals. Some siderophores or killer toxins produced by marine yeasts have ability to inhibit growth of pathogenic bacteria or kill pathogenic yeasts in marine animals. The virulent factors from marine pathogens can be genetically displayed on marine yeast cells, and the yeast cells displaying the virulent factors can stimulate marine animals to produce specific antibody against the pathogens. Some marine yeast cells are rich in proteins and essential amino acids and can be used in nutrition for marine animals. The marine yeast cells rich in lipid can be used for biodiesel production. Recently, it has been reported that some strains of Yarrowia lipolytica isolated from marine environments can produce nanoparticles. Because many marine yeasts can remove organic pollutants and heavy metals, they can be applied to remediation of marine environments. It has been shown that the enzymes produced by some marine yeasts have many unique properties and many potential applications.     

Ashbya gossypii: a model for fungal developmental biology

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence.     

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as ‘complementary’ stress-targets

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25–150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.     

A selective medium for detecting yeasts capable of spoiling wine

By careful manipulation of nutrients, pH, and ethanol concentration, a medium has been developed which permits the growth of yeasts capable of spoiling table wines yet restricts growth of other contaminants. The selectivity of the broth was evaluated using yeasts which were known to spoil, or not spoil, a variety of table wines. All of the yeasts tested that were capable of spoiling wine grew in the broth within 72 h. Over 77% of the non-spoilers failed to grow in the broth during the 3 d incubation.
A test, using this broth, has been introduced into several wine-bottling plants in the UK, and is proving to be a useful predictor of yeast spoilage. A pre-enrichment medium has been formulated which ensures that stressed cells are also detected. Automation of the test, by exploiting a robotic arm and suitable photometry, is discussed. If desired, a quicker detection of wine-spoiling yeasts can be achieved by using the medium in conjunction with rapid methods such as ATP-assay.     

Cytotoxic mechanism of selenomethionine in yeast

Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells.     

Effects of live Saccharomyces cerevisiae cells on zoospore germination,growth, and cellulolytic activity of the rumen anaerobic fungus,Neocallimastix frontalis MCH3

The effects of a live yeast strain of Saccharomyces cerevisiae have been investigated on zoospore germination, metabolism, and cellulolytic activity of the anaerobic rumen fungus Neocallimastix frontalis MCH3. The addition of yeast cells to a vitamin-deficient medium stimulated the germination of fungal zoospores, increased cellulose degradation and hydrogen, formate, lactate, and acetate production. Responses depended on the concentration of yeast cells added and on their viability. Yeast supplementation provided vitamins such as thiamine, which is essential for fungal growth and activity. These results demonstrate that yeasts could enhance plant cell wall colonization by N. frontalis. With certain diets, yeasts could therefore be a good tool to optimize the microbial degradation of lignocellulosic materials, but more research is needed to understand their mechanisms of action, so that they can be used with maximum efficiency as feed supplements.     

Yeast entrapment in Duckweeds: Immobilization performance and biomass localization within the support particles

Cell structured support material (CSM) prepared from Wolffia arrhiza fronds were loaded with Saccharomyces cereevisiae by the propagation of yeast cells introduced in the aerenchyma. The loading process could be strongly accelerated by preventing the growth of freely suspended yeasts in the fermenter. The pathway of the inoculation through the stomata could be visualized by transparent light microscopy of amylase and protease treated CSM. Slices of the loaded CSM clearly showed that the framework of the inner chlorenchyma cells remains intact during the propagation of the included yeast cells. Most of the yeast cells were found to be localized as a dense suspension within unbroken cells. During long-term treatment of the loaded CSM at high convection, about one third of the yeasts was released into water with a constant rate greater than 0.34 h^?1. The residual amount of yeast cells remained in a stable compartment from which further loss did not occur for the period tested (20–48 h). It is assumed that the stable compartment is identical with the interior of chlorenchyma cells. The results are discussed in relationship to the possible use of the immobilized biomass in fermentation.     

Yeasts from high-altitude lakes: influence of UV radiation

Mountain lakes located at a high elevation are typically exposed to high UV radiation (UVR). Little is known about the ecology and diversity of yeasts inhabiting these extreme environments. We studied yeast occurrence (with special emphasis on those producing carotenoid pigments) at five high-altitude (>1400 m a.s.l.) water bodies located in the Nahuel Huapi National Park (Bariloche, Argentina). Isolates were identified using a polyphasic approach. Production of photoprotective compounds (carotenoids and mycosporines) by yeast isolates, and UVB resistance of selected species were studied. All water samples contained viable yeast cells in variable numbers, generally ranging from 49 to 209 cells L⁻¹. A total of 24 yeast species was found; at least four represented novel species. Carotenogenic yeasts prevailed in lakes with low water conductivity and higher transparency and chlorophyll a levels. Apparently, the ability to produce photoprotective compounds in yeasts was related to the transparency of mountain lake waters, and strains from more transparent waters developed increased UVB resistance. Our results indicate that UVR is an important environmental factor affecting the yeast community structure in aquatic habitats.     

Use of n-hexadecane as an oxygen vector to improve Phaffia rhodozyma growth and carotenoid production in shake-flask cultures

AIMS: To identify beneficial oxygen vectors for Phaffia rhodozyma in liquid cultures, and to evaluate their use to improve the oxygen transfer and carotenoid production in the yeast cultures. METHODS AND RESULTS: Several liquid hydrocarbons were tested as oxygen vectors for improving the yeast growth and carotenoid production in shake-flask cultures of P. rhodozyma. While all nontoxic organic liquids (Log P: > or =5.6) showed a positive effect, n-hexadecane was proved to be the most beneficial for the yeast growth and carotenoid production. The addition of 9% (v/v) n-hexadecane to the liquid medium at the time of inoculation was found to be optimal, increasing the carotenoid yield by 58% (14.5 mg l(-1) vs 9.2 g l(-1) in the control) and the oxygen transfer rate (OTR) by 90%. CONCLUSIONS: The addition of n-hexadecane to shake-flask cultures of P. rhodozyma significantly improved the oxygen transfer in culture, thus increasing the carotenoid production. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of organic oxygen vectors such as n-hexadecane may be a simple and useful means for enhancing oxygen transfer and carotenoid production in liquid fermentation of P. rhodozyma.     

Modelling Biolog profiles' evolution for yeast growth monitoring in alcoholic fermentation

Aims: A research was undertaken to explore the possibility to express with suitable mathematical models Biolog metabolic curves obtained for oenological yeasts and to use such models for monitoring yeast growth in alcoholic fermentation. Methods and Results: Experimental curves of metabolic activity in Biolog YT microplates, obtained in a previous work for various oenological yeast strains in pure cultures and mixed populations, at various cell concentrations, have been modelled with Gompertz’s, Gompertz’s modified and Lindstrom’s mathematical equations. Lindstrom’s model proved to be the most suitable to fit the curves of the oenological yeasts under study, providing the highest correlation coefficients between experimental and calculated data. The model made it possible to recognize, in mixed yeast populations, the presence of active dry yeasts used for guided fermentations. Model’s constant parameters were used for a numerical characterization of yeast curves. Conclusions: The application of the model to the experimental data resulted to be suitable for an early prediction of the successive evolution of yeast growth. Significance and Impact of the Study: The results obtained indicate the possibility to develop protocols for monitoring yeast presence during alcoholic fermentation, with an early assessment of the correct evolution of their growth, especially when active dry yeasts are employed.     

Respiratory instability in distillery yeasts

Summary Eight distillery yeasts and one haploid strain of Saccharomyces cerevisiae were screened for the presence of respiratory deficient cells in their populations after culturing in either yeast extract-peptone-sucrose medium or in molasses medium. It was found that the distillery yeasts yield RD cells similar to the haploid with the number varying with the yeast strain and growth temperature. In cell biomass recycling, the number of RD cells also increases considerably.     

Indole-3-Acetic Acid-Producing Yeasts in the Phyllosphere of the Carnivorous Plant Drosera indica L

Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.