Yeast Torulaspora Research Articles

Influence of Fermentation Time and Inoculation of Starter Culture on the Chemical Composition of Fermented Natural Coffee Followed by Depulping.

Fermentation using starter cultures has been considered an alternative and economically viable technology for the production of specialty coffees. This type of technology promotes several benefits, such as increased sensory quality, control over the fermentation process, predictability of the final product and added value. Coffee (Coffea arabica L.) samples for this study were collected in Presidente Olegário - MG (2018/19 crop year) in the Cerrado region of Minas Gerais. The effects of natural fermentation and inoculation of the yeast Torulaspora delbrueckii and duration of fermentation (0, 24, 48, 72 and 96 hours) on the sensory and chemical quality (analysis of bioactive, volatile, and organic compounds and fatty acids) of coffee were evaluated. The objective of this study was to determine the effect of fermentation time and starter culture inoculation on the chemical composition of fermented coffees. Fermentation time significantly influenced the sensory description of the coffee beverage, with notes of honey, brown sugar and almond predominating up to 48 hours, for coffees fermented for 72 and 96 hours the notes described were and fruity, winey notes. The chemical composition was primarily influenced by fermentation time.

Natural fermentation with delayed inoculation of the yeast Torulaspora delbrueckii: Impact on the chemical composition and sensory profile of natural coffee

All coffee production stages occur in a microbiome, which is generally composed of bacteria, yeasts, and filamentous fungi. The use of starter cultures in post-harvest processing stages is an interesting alternative, since they promote faster removal of mucilage and incorporation of compounds that improve sensory quality, which can result in diverse sensory attributes for the beverage. This study was therefore developed with the objective of evaluating the effect of the following processing procedures on the chemical and sensory characteristics of the coffee beverage: first, fermentation of coffee fruit of the yellow Catucaí variety of Coffea arabica with indigenous microorganisms, followed by inoculation of the starter culture Torulaspora delbrueckii CCMA 0684 during the drying stage. The fruit was divided into two lots, which were differentiated by a natural fermentation process before drying began. The starter culture was inoculated on the coffee at different times during the drying process: at 0 h, 24 h, 48 h, or 72 h after drying began. The sensory attributes, the volatile compound composition of the roasted beans, the organic acid profile, the bioactive compounds, and the fatty acid profile of the green coffee beans were analyzed. The fatty acid and bioactive compound content showed little variation among treatments. Analysis of volatile compounds and organic acids and evaluation of sensory attributes made it possible to distinguish the two treatments. We conclude that natural fermentation of coffee fruit improve the chemical and sensory quality of the coffee beverage. The effect of natural fermentation may be before inoculation of the starter cultures or even during drying.

Maize treatment with yeast cells induces resistance against Fusarium rot.

Maize is one of the most cultivated species and requires agrochOPemicals for nutrition and pathogen control. Fusarium verticillioides is responsible for damaging plants and stored grains. Plants naturally exposed to stresses have defense mechanisms that are triggered by chemical or biological agents, known as induced systemic resistance. In this study, the yeast Torulaspora globosa (strain CCA6S01) was evaluated as an immune response promoter in maize against F. verticillioides. The treatments started 4 days after maize emergence and consisted of control (saline solution), acetylsalicylic acid (ASA, 100 mg/L), yeast cells (1×105 cells/mL), or yeast metabolites (cell-free filtrates). After running the treatments, the plants were inoculated with 1 mL of a F. verticillioides suspension at a concentration of 1×106 spores/mL. The application of yeast cells provided similar results to ASA treatment, a known inducer of plant resistance. Yeast cells provided maize plants with fewer rot symptoms and higher activities of enzymes related to plant resistance. Thus, we concluded that T. globosa (strain CCA6S01) might be used in agriculture practice as a plant protection agent. It can help to decrease the application of fungicides in the field and maintain plant productivity under stress.

Draft Genome Sequence of the Yeast Torulaspora quercuum Strain UCD657, Isolated from Soil in Ireland.

Torulaspora quercuum is an ascomycete yeast first isolated in 2009. Here, we present the genome sequence of T. quercuum isolate UCD657, which was isolated from soil in Ireland. This genome is 10.4 Mb and was assembled into 8 chromosome-sized scaffolds of >1 Mb in size, plus a mitochondrial genome scaffold.

The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein.

To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a nonduplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1 at the silencer. Here, we examined whether the emergence of Sir1 coincided with Orc1 acting as a silencer-binding protein. In the nonduplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC-binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae.

Exploring the feasibility of biotransforming salted soy whey into a soy sauce-like condiment using wine yeast Torulaspora delbrueckii and soy sauce yeasts Zygosaccharomyces rouxii and Candida versatilis as single starter cultures

Salted soy whey, a liquid by-product from salted tofu processing, is a source of valuable nutrients. However, it is often under-utilized due to its high salt content. The objective of this study was to investigate the feasibility of using different species of yeast to transform salted soy whey into a soy sauce-like condiment. Three yeasts were used for salted soy whey biotransformation: Zygosaccharomyces rouxii NCYC 1682, Candida versatilis NCYC 1433, and Torulaspora delbrueckii Biodiva. This study focused on the growth of the yeasts in soy whey added with different levels of NaCl and the physicochemical changes of salted soy whey after fermentation. The soy sauce yeasts (Z. rouxii and C. versatilis) grew by approximately 2 log CFU/mL in soy whey with 2% and 10% salt while the cell count of wine yeast T. delbrueckii increased by around 1.5 log CFU/mL at 10% salt and 1.78 log CFU/mL at 2% salt after 14 days of fermentation. Candida versatilis grew better at 10% salt with less glucose consumption (consumed 68.49% at 10% versus 94.05% at 2% salt) than at 2% salt. It was also found that all three yeasts converted isoflavone glycosides (bound isoflavones) into aglycones (free isoflavones), the latter having better bioavailability. C. versatilis showed the greatest ability to transform isoflavone glycosides in salted soy whey into higher amounts of aglycones (conversion efficiency of 23.04% at 2% salt and 15.05% at 10% salt). Salted soy whey fermented with different yeasts had different volatile profiles. Soy sauce yeasts produced more isobutyl alcohol, isoamyl alcohol and volatile phenols while T. delbrueckii generated a substantial amount of ethanol and esters. This study revealed the growth and flavour modulating potential of yeasts in salted soy whey fermentation, which provides a possible avenue to develop a soy sauce-like condiment using salted soy whey as a substrate.

A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast.

Microbe domestication has a major applied relevance but is still poorly understood from an evolutionary perspective. The yeast Torulaspora delbrueckii is gaining importance for biotechnology but little is known about its population structure, variation in gene content or possible domestication routes. Here, we show that T.delbrueckii is composed of five major clades. Among the three European clades, a lineage associated with the wild arboreal niche is sister to the two other lineages that are linked to anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix-Anthropic clade). Using 64genomes we assembled the pangenome and the variable genome of T.delbrueckii. A comparison with Saccharomyces cerevisiae indicated that the weight of the variable genome in the pangenome of T.delbrueckii is considerably smaller. An association of gene content and ecology supported the hypothesis that the Mix-Anthropic clade has the most specialized genome and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. In contrast to S.cerevisiae, domestication in T.delbrueckii is not primarily driven by alcoholic fermentation but rather by adaptation to dairy and bread-production niches. This study expands our views on the processes of microbe domestication and on the trajectories leading to adaptation to anthropic niches.

Yeast Plasma Membrane Fungal Oligopeptide Transporters Display Distinct Substrate Preferences despite Their High Sequence Identity.

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.

Physiological, Biochemical and Energetic Characteristics of Torulaspora globosa, a Potential Producer of Biofuel

It was obtained that the yeast Torulaspora globosa VKPM Y-953 is suitable for the production of biodiesel fuel. Zinc plays an important regulatory role in the metabolism of the studied strain. The study of the growth parameters and the fatty acid profile of the yeast T. globosa showed that the limitation of its growth by ethanol, at different concentrations of zinc in the medium, considerably influences the chemical composition and the energy content of yeast cells, but not their yield by weight (Yx/s). The increased concentrations of zinc in the medium, in combination with the yeast growth limitation by ethanol, elevated the content of lipids in the cells by 28% and diminished the content of proteins by 14%. At the same time, the limitation of yeast growth by zinc decreased Yx/s and energy (ηX/S) by 2.6 and 3.1 times, respectively. In this case, the content of lipids in cells fell by 72%, while that of proteins rose by 65%. The fatty acid profile of the T. globosa cells was used to estimate the main characteristics of biodiesel (iodine value, cetane number, density, and kinematic viscosity). The biomass of T. globosa can also be used in agriculture as a feed additive rich in essential amino acids.

Effect of single amino acid addition on growth kinetics and flavor modulation by Torulaspora delbrueckii in soy (tofu) whey alcoholic beverage fermentation

Soy (tofu) whey is a by-product commonly disposed of by tofu manufacturers around the world. Due to its nutritious nature, direct disposal of soy whey into the sewage can result in a detrimental impact on the environment in the long-run. In this study, soy whey supplemented with four individual amino acids (valine, leucine, isoleucine and phenylalanine) equivalent to 120 mg N/L was fermented with a yeast Torulaspora delbrueckii Biodiva. The supplementation of an individual amino acid resulted in faster sugar utilization and lower levels of residual sugar than the unsupplemented whey but did not result in a significantly higher amount of ethanol (7–8% v/v) at the end of fermentation. Isoleucine supplementation resulted in a slightly slower initial yeast growth rate when compared to the control while the other three amino acids had identical yeast growth kinetics to the control. Isoleucine supplementation also resulted in slower sugar utilization during the first four days. Therefore, isoleucine is least preferred by the yeast. The supplementation of amino acids resulted in greater formation of higher alcohols and their corresponding alcohol-derived esters. Overall, the supplementation of a single amino acid enhanced sugar utilization and impacted flavor compounds of the resultant soy whey alcoholic beverage.

A white-to-opaque-like phenotypic switch in the yeast Torulaspora microellipsoides

Torulaspora microellipsoides is an under-characterized budding yeast of the Saccharomycetaceae family that is primarily associated with viticulture. Here we report for the first time to our knowledge that T. microellipsoides undergoes a low-frequency morphological switch from small budding haploid (white) yeast to larger, higher ploidy (opaque) yeast. Comparison of transcriptomes by mRNA-seq revealed 511 differentially regulated genes, with white cells having greater expression of genes involved in stress resistance and complex carbohydrate utilization, and opaque cells up-regulating genes involved in ribosome biogenesis. Growth assays showed that white cells are physiologically more resistant to stationary-phase conditions and oxidative stress, whereas opaque cells exhibited greater cold tolerance. We propose that phenotypic switching in T. microellipsoides is an ecological adaptation, as has been suggested for similar morphological switching in distantly related species like Candida albicans, and we propose that this switching is a more broadly utilized biological strategy among yeasts than previously thought.

Biocatalytic Synthesis of Natural Dihydrocoumarin by Microbial Reduction of Coumarin

Dihydrocoumarin is a natural product of great relevance for the flavour industry. In this work, we describe a study on the biotransformation of the toxic compound coumarin into natural dihydrocoumarin, recognized as safe for food aromatization. To this end, we screened a variety of yeasts and filamentous fungi, isolated from different sources, in order to evaluate their ability to reduce selectively the conjugated double bond of coumarin. Moreover, since coumarin induces cytotoxicity and therefore inhibits cell growth as well as the cell metabolic activity, we tested out different substrate concentrations. All strains were able to convert the substrate, although showing very different conversion rates and different sensitivity to the coumarin concentration. In particular, the yeasts Torulaspora delbrueckii, Kluyveromyces marxianus and the fungus Penicillium camemberti displayed the higher activity and selectivity in the substrate transformation. Among the latter strains, Kluyveromyces marxianus presented the best resistance to substrate toxicity, allowing the biotransformation process even with coumarin concentration up to 1.8 g/L.

Finite element model to study potassium dynamics in the rhizosphere of a wheat root due to presence of bio-physical source

Potassium is a crucial nutrient required for maintaining the health of a plant. It increases the resistance power of the plant against the diseases. Apart from this potassium plays important role in various other physical and physiological functions involved in the growth of a plant. Uptake of potassium by the root of crop plant depends upon its availability in the rhizosphere of the root. The potassium dynamics in the soil is not well understood till date. In this paper an attempt has been made to study the potassium dynamics in the rhizosphere of the root of wheat crop in the presence and absence of bio-physical source. A finite element model is proposed for a two dimensional unsteady state case. A bio-physical source consisting of rock powder and yeast torulaspora globosa is assumed to be present in the soil. The rock powder contains insoluble potassium which is converted into soluble form of potassium by yeast torulaspora globosa. The model incorporates the term water flux, diffusion coefficient, soil porosity, soil buffer power, uptake coefficient of root etc. The results have been computed using MATLAB. The numerical results have been used to analyze the impact of bio-physical source on the potassium dynamics in the rhizosphere of a root of wheat. The presence of bio-fertilizer leads to significant changes in potassium concentration in the rhizosphere of the wheat root and therefore the bio-physical source consisting of rock powder and yeast torulaspora globosa is recommended as a bio-fertilizer for maintenance of health and growth of wheat crop.

Effect of selenium on growth and antioxidant enzyme activities of wine related yeasts.

The use of supplements in the diet is a common practice to address nutritional deficiencies. Selenium is an essential micronutrient with an antioxidant and anti-carcinogenic role in human and animal health. There is increasing interest in developing nutritional supplements such as yeast cells enriched with selenium. The possibility of producing beverages, namely wine, with selenium-enriched yeasts, led us to investigate the selenium tolerance of six wine related yeasts. The production of such cells may hamper selenium toxicity problems. Above certain concentrations selenium can be toxic inducing oxidative stress and yeast species can show different tolerance. This work aimed at studying selenium tolerance of a diversity of wine related yeasts, thus antioxidant response mechanisms with different concentrations of sodium selenite were evaluated. Viability assays demonstrated that the yeast Torulaspora delbrueckii showed the highest tolerance for the tested levels of 100 µg mL(-1) of sodium selenite. The evaluation of antioxidative enzyme activities showed the best performance for concentrations of 250 and 100 µg mL(-1), respectively for the yeast species Saccharomyces cerevisiae and Hanseniaspora guilliermondii. These results encourage future studies on the possibility to use pre-enriched yeast cells as selenium supplement in wine production.

Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast Torulaspora delbrueckii.

The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼1900 years ago), and to the Neolithic era for bioprocesses (∼4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.

Release of potassium from rock powder by the yeast Torulaspora globosa

The alteration of minerals in rocks and the availability of elements for plant nutrition require long periods of time, and microorganisms are thought to induce the release of potassium and phosphate from rocks. In this context, this work evaluates the role of the yeast Torulaspora globosa, isolated from the sugar cane rhizosphere, in the solubilization of potassium from alkaline ultramafic rock powder. The experiments were performed in liquid medium, with or without agitation, at 30°C with the following treatments: culture medium + alkaline ultramafic; culture medium + yeast suspension; and culture medium + yeast suspension + alkaline ultramafic. The results showed that as much as 38% of the total potassium in the rock was released in the medium with the yeast during a 15-day period of incubation. Acid production may be the mechanism by which the yeast solubilizes potassium because the total acidity increased during the sampling period. Agitation (which increased oxygen availability) resulted in approximately 20% more biosolubilization of the alkaline ultramafic rock than with the static culture. These data indicate the potential for this yeast in biosolubilization processes and biofertilizer production.

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41-293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP-binding region (positions 48-71) and a consensus Ser/Thr protein kinase active site (positions 160-172).

Evaluation of the biological control by the yeast Torulaspora globosa against Colletotrichum sublineolum in sorghum

The yeasts are microorganisms with great potential for biotechnological applications in diverse areas. The biological control of phytopathogens by yeasts has showed satisfactory results under laboratory conditions, and it has already produced commercial formulations. With this as focus, this work aims to perform in vitro and in vivo evaluations of the action of a Torulaspora globosa yeast strain (1S112), isolated from sugarcane rhizosphere, against the phytopathogenic mold Colletotrichum sublineolum, the causative agent of anthracnose in sorghum. In vitro experiments included the antagonism test in Petri dishes with morphological hyphal evaluation; yeast killer activity; siderophore, volatile compound and hydrolytic enzyme production. In vivo experiments were conducted in greenhouse conditions with a sorghum variety susceptible to C. sublineolum by evaluating the anthracnose disease for 6 weeks. The results indicated that the yeast strain significantly controlled the fungal growth, either in vitro or in vivo. The strain of T. globosa exhibited killer activity against two sensitive strains, which is a novel capacity for this species. The yeast did not produce siderophores, volatile compounds or hydrolytic enzymes, although it has reduced the mycelial growth, resulting in hyphal deformities but not cell death. The yeast controlled the anthracnose disease in sorghum, either inoculated before or after the fungal spores, suggesting that the competition for space and nutrients to dominate the mold and killer toxin production, altering the hyphal morphology, are mechanisms utilized by the yeast in the biocontrol.

Influence of Antimicrobial Agents on the Spoilage of a Meat-Based Entomophage Diet

The microbial decomposition of a meat-based entomophage diet presented in Parafilm packets was investigated. Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hens' eggs, liver, and ground beef). At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations. Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24 h (approximately 10(10) colony-forming units per gram). Bacterial populations also increased in diets containing antibacterial agents, but they were significantly smaller than in diets not containing antibacterial agents. The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used. The proliferation of fungi was delayed relative to bacteria, but significant differences were observed among the diet treatments. Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents. The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica. The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi. The results of this study demonstrate that antimicrobial agents significantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in Parafilm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species.

The relative glucose uptake abilities of non-Saccharomyces yeasts play a role in their coexistence with Saccharomyces cerevisiae in mixed cultures.

The growth and glucose uptake of single cultures of the wine-related yeasts Kluyveromyces thermotolerans, Torulaspora delbrueckii, and Saccharomyces cerevisiae were investigated. The yeasts had different specific glucose uptake rates (qs) that depended on the residual glucose concentration and the oxygen availability. In mixed cultures, the qs values of the yeasts were not subject to any interaction effects over a wide range of glucose concentrations. Our results strongly indicate that the relative glucose uptake abilities of both non-Saccharomyces yeasts, i.e. the qs(non-Saccharomyces)/qs(S. cerevisiae) ratios, regulated their abilities to compete for space in mixed cultures with S. cerevisiae, which, in turn, regulated their early deaths. This hypothesis enabled us to explain why K. thermotolerans was less able than T. delbrueckii to coexist with S. cerevisiae in mixed cultures. Furthermore, it enabled us to explain why oxygen increased the abilities of K. thermotolerans and T. delbrueckii to coexist with S. cerevisiae in the mixed cultures.