Cerevisiae Isolates Research Articles

The bacterial and yeast microbiota in livestock forages in Hungary

BackgroundAlong bacteria, yeasts are common in forages and forage fermentations as spoilage microbes or as additives, yet few studies exist with species-level data on these fungi’s occurrence in feedstuff. Active dry yeast and other yeast-based products are also common feed additives in animal husbandry. Here, we aimed to characterize both fermented and non-fermented milking cow feedstuff samples from Hungary to assess their microbial diversity in the first such study from Central Europe.ResultsWe applied long-read bacterial metabarcoding to 10 fermented and 25 non-fermented types of samples to assess bacterial communities and their characteristics, surveyed culturable mold and yeast abundance, and identified culturable yeast species. Fermented forages showed the abundance of Aerococcaceae, Bacillaceae, Brucellaceae, Lactobacillaceae, Staphylococcaceae, and Thermoactinomycetaceae, non-fermented ones had Cyanothecaceae, Enterobacteriaceae, Erwiniaceae, Gomontiellaceae, Oxalobacteraceae, Rhodobiaceae, Rickettsiaceae, and Staphylococcaceae. Abundances of bacterial families showed mostly weak correlation with yeast CFU numbers, only Microcoleaceae (positive) and Enterococcaceae and Alcaligenaceae (negative correlation) showed moderate correlation. We identified 14 yeast species, most commonly Diutina rugosa, Pichia fermentans, P. kudriavzevii, and Wickerhahomyces anomalus. We recorded S. cerevisiae isolates only from animal feed mixes with added active dry yeast, while the species was completely absent from fermented forages. The S. cerevisiae isolates showed high genetic uniformity.ConclusionOur results show that both fermented and non-fermented forages harbor diverse bacterial microbiota, with higher alpha diversity in the latter. The bacterial microbiome had an overall weak correlation with yeast abundance, but yeasts were present in the majority of the samples, including four new records for forages as a habitat for yeasts. Yeasts in forages mostly represented common species including opportunistic pathogens, along with a single strain of Saccharomyces used as a feed mix additive.

Graph-Based Pan-Genome Reveals the Pattern of Deleterious Mutations during the Domestication of Saccharomyces cerevisiae.

The "cost of domestication" hypothesis suggests that the domestication of wild species increases the number, frequency, and/or proportion of deleterious genetic variants, potentially reducing their fitness in the wild. While extensively studied in domesticated species, this phenomenon remains understudied in fungi. Here, we used Saccharomyces cerevisiae, the world's oldest domesticated fungus, as a model to investigate the genomic characteristics of deleterious variants arising from fungal domestication. Employing a graph-based pan-genome approach, we identified 1,297,761 single nucleotide polymorphisms (SNPs), 278,147 insertion/deletion events (indels; <30 bp), and 19,967 non-redundant structural variants (SVs; ≥30 bp) across 687 S. cerevisiae isolates. Comparing these variants with synonymous SNPs (sSNPs) as neutral controls, we found that the majority of the derived nonsynonymous SNPs (nSNPs), indels, and SVs were deleterious. Heterozygosity was positively correlated with the impact of deleterious SNPs, suggesting a role of genetic diversity in mitigating their effects. The domesticated isolates exhibited a higher additive burden of deleterious SNPs (dSNPs) than the wild isolates, but a lower burden of indels and SVs. Moreover, the domesticated S. cerevisiae showed reduced rates of adaptive evolution relative to the wild S. cerevisiae. In summary, deleterious variants tend to be heterozygous, which may mitigate their harmful effects, but they also constrain breeding potential. Addressing deleterious alleles and minimizing the genetic load are crucial considerations for future S. cerevisiae breeding efforts.

Selection of indigenous Saccharomyces cerevisiae strains with good oenological and aroma characteristics for winemaking in Ningxia China

Ningxia is one of the well-known wine producing regions in China. However, the oenological and aroma characteristics of indigenous yeasts remains hidden. The fermentative and oenological properties including stress resistance, hydrogen sulfide, foam production levels; killer phenotype, and flocculation of 89 Ningxia indigenous Saccharomyces cerevisiae isolates and ten commercial yeasts were evaluated. The fermentative and oenological properties of the tested strains varied significantly. They could resist 500 g/L glucose, 300 mg/L SO2, 14% (v/v) ethanol and pH 2.8, and produce more esters. They also produce low levels of ethanol and could conduct fermentations vigorously and at a high rate. Cabernet Sauvignon wines made with NXU 21–24 showed the high intensity of tropical fruit, dry fruit, temperate fruit, and spicy flavor. The floral flavor in NXU 21–102 fermented wine is very intense. The indigenous S. cerevisiae strains of NXU 21–102 and NXU 21–24 exhibited potential use as starter cultures in wine production.

Wild yeast isolation by middle school students reveals features of North American oak populations of Saccharomyces cerevisiae and Kluyveromyces lactis.

Features of the natural life cycle of the budding yeast Saccharomyces cerevisiae were crucial to its domestication as a laboratory experimental model, especially the ability to maintain stable haploid clones and cross them at will to combine alleles via meiosis. Stable haploidy results from mutations in HO, which encodes an endonuclease required for haploid-specific mating-type switching. Previous studies found an unexpected diversity of HO alleles among natural isolates within a small geographic area. We developed a hands-on field and laboratory activity for middle school students in Denver, Colorado, USA to isolate wild yeast from oak bark, identify species via DNA sequencing, and sequence HO from S. cerevisiae isolates. We find limited HO diversity in North American oak isolates, pointing to efficient, continuous dispersal across the continent. By contrast, we isolated the "dairy yeast", Kluyveromyces lactis, from a tree <10 m away and found that it represents a new population distinct from an oak population in an adjacent state, pointing to high genetic diversity. The outreach activity partnered middle school, high school, and university students in making scientific discoveries and can be adapted to other locations and natural yeast habitats. Indeed, a pilot sampling activity in southeast Texas yielded S. cerevisiae oak isolates with a new allele of HO and, from a nearby prickly pear cactus, a heat-tolerant isolate of Saccharomyces paradoxus.

Differentiation of Saccharomyces boulardii from Saccharomyces cerevisiae Strains Using the qPCR-HRM Technique Targeting AAD15 Gene

ABSTRACT S. boulardii, the probiotic variety of S. cerevisiae, is known for its different characteristics, even though the two strains share more than 99% genomic relatedness. Therefore, S. boulardii has been examined and evaluated for differential characteristics in many studies. This study aims to explain the differentiation of S. boulardii from well-identified S. cerevisiae strains by biochemical characteristics and the qPCR-HRM. Initially, S. cerevisiae isolates from the HUF Culture Collection, were screened for basic biochemical characteristics such as growth temperature, glucose, and galactose utilization tests. Out of all selected cultures, the viability at gastric pH was investigated by spectrophotometric readings. The results showed that one isolate was more resistant than other strains when exposed to gastric acids. After sequencing and phylogeny steps using LR0R universal primer, there was no significant differentiation for S. boulardii strains and therefore HRM analysis was performed to interrogate sequence variation and differentiate S. boulardii strains. HRM analysis has successfully distinguished even highly closely related yeast strains.

Effects of the coculture of Pediococcus pentosaceus and Saccharomyces cerevisiae with different leavening ability on the quality and volatile organic compounds of Chinese steamed bread

Pediococcus pentosaceus has a promising potential to improve the quality and flavor of Chinese steamed bread (CSB), but its synergistic effect with Saccharomyces cerevisiae is still unclear. In this study, P. pentosaceus was cocultured with three S. cerevisiae isolates separately, which had different leavening ability. The times required for the fermentation of dough by 2 times the volume of S. cerevisiae isolates ACX 0089, ACX 0490 and ACX 0078 were 46, 88 and 120 min, respectively. The results showed that the leavening ability of S. cerevisiae had a significant impact on the quality of CSBs. Compared other S. cerevisiae isolates, the ACX 0490 cocultured with P. pentosaceus increased the contents of bound water and free water in CSB, decreased the content of semibound water, and produced larger and regular porosity, thus endowed a greater specific volume and softer texture. A total of 36 volatile organic compounds (VOCs) were found in CSBs, with alcohols accounting for the main component. The coculture of P. pentosaceus with the intermediate leavening ability of S. cerevisiae ACX 0490 produced the highest proportion of esters among all CSBs. Moreover, S. cerevisiae ACX 0490 endowed the CSB unique VOCs, including cyclodecanol, ethylacetate, and 3,5-dimethylbenzaldehyde. The results of sensory evaluation also showed that S. cerevisiae ACX0490 cocultured CSB obtained the highest scores among all the test samples. The results provided a reference for selecting S. cerevisiae isolates with different leavening ability and the combination of P. pentosaceus to achieve the best quality and flavor of CSB.

Tannin-Tolerant Saccharomyces cerevisiae Isolated from Traditional Fermented Tea Leaf (Miang) and Application in Fruit Wine Fermentation Using Longan Juice Mixed with Seed Extract as Substrate.

This study focused on isolating tannin-tolerant yeasts from Miang, a fermented tea leaf product collected from northern Laos PDR, and investigating related food applications. From 43 Miang samples, six yeast isolates capable of ethanol production were obtained, with five isolates showing growth on YPD agar containing 4% (w/v) tannic acid. Molecular identification revealed three isolates as Saccharomyces cerevisiae (B5-1, B5-2, and C6-3), along with Candida tropicalis and Kazachstania humilis. Due to safety considerations, only Saccharomyces spp. were selected for further tannic acid tolerance study to advance food applications. Tannic acid at 1% (w/v) significantly influenced ethanol fermentation in all S. cerevisiae isolates. Notably, B5-2 and C6-3 showed high ethanol fermentation efficiency (2.5% w/v), while others were strongly inhibited. The application of tannin-tolerant yeasts in longan fruit wine (LFW) fermentation with longan seed extract (LSE) supplementation as a source of tannin revealed that C6-3 had the best efficacy for LFW fermentation. C6-3 showed promising efficacy, particularly with LSE supplementation, enhancing phenolic compounds, antioxidant activity, and inhibiting α-glucosidase activity, indicating potential antidiabetic properties. These findings underscore the potential of tannin-tolerant S. cerevisiae C6-3 for fermenting beverages from tannin-rich substrates like LSE, with implications for functional foods and nutraceuticals promoting health benefits.

Preliminary Study on Yeasts Associated with the Production of "Tostado"-a Traditional Sweet Wine from Galicia (NW Spain).

Tostado is a traditional sweet wine from the Designations of Origins (DOs) of Ribeiro and Valdeorras in Galicia (NW Spain). The harvested grapes are air-dried and pressed to increase the concentrations of sugars, acids, and flavour compounds. Therefore, knowledge of the yeasts involved in fermentation under these conditions is essential to guarantee the quality and uniqueness of the valuable, distinctive, and expensive Tostado wines. Saccharomyces and non-Saccharomyces yeasts were identified using Wallerstein Laboratory (WL) Nutrient Agar and lysine plating, followed by polymerase chain reaction (PCR) amplification, enzymatic digestion, and sequencing. Saccharomyces cerevisiae isolates were further characterised at the strain level using mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP). Statistical analyses were also performed, including different diversity indices, Similarity Percentage (SIMPER) analysis, principal component analysis (PCA), neighbor-joining clustering, parsimony-phylogram, and network plot. In addition, the total acidity, volatile acidity, reducing sugars, and alcoholic strength by volume of the Tostado wines were analysed. A wide diversity of autochthonous yeasts was found, which were predominantly species of oenological relevance, such as Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, Debaryomyces hansenii, Torulaspora delbrueckii, Pichia spp., and Saccharomyces cerevisiae from the must and paste stages of Tostado wine. In addition, 19 different S. cerevisiae strains were identified. This high yeast diversity, which changed from the early stages of fermentation, could contribute to the distinctive characteristics observed in Tostado wine. Characteristic and differentiating chemical and microbiological profiles were found as early as the pre-fermentation stages, which adds value to these special wines that have rarely been studied.

Ancient and recent origins of shared polymorphisms in yeast.

Shared genetic polymorphisms between populations and species can be ascribed to ancestral variation or to more recent gene flow. Here, we mapped shared polymorphisms in Saccharomyces cerevisiae and its sister species Saccharomyces paradoxus, which diverged 4-6 million years ago. We used a dense map of single-nucleotide diagnostic markers (mean distance 15.6 base pairs) in 1,673 sequenced S. cerevisiae isolates to catalogue 3,852 sequence blocks (≥5 consecutive markers) introgressed from S. paradoxus, with most being recent and clade-specific. The highly diverged wild Chinese S. cerevisiae lineages were depleted of introgressed blocks but retained an excess of individual ancestral polymorphisms derived from incomplete lineage sorting, perhaps due to less dramatic population bottlenecks. In the non-Chinese S. cerevisiae lineages, we inferred major hybridization events and detected cases of overlapping introgressed blocks across distinct clades due to either shared histories or convergent evolution. We experimentally engineered, in otherwise isogenic backgrounds, the introgressed PAD1-FDC1 gene pair that independently arose in two S. cerevisiae clades and revealed that it increases resistance against diverse antifungal drugs. Overall, our study retraces the histories of divergence and secondary contacts across S. cerevisiae and S. paradoxus populations and unveils a functional outcome.

Diallel panel reveals a significant impact of low-frequency genetic variants on gene expression variation in yeast.

Unraveling the genetic sources of gene expression variation is essential to better understand the origins of phenotypic diversity in natural populations. Genome-wide association studies identified thousands of variants involved in gene expression variation, however, variants detected only explain part of the heritability. In fact, variants such as low-frequency and structural variants (SVs) are poorly captured in association studies. To assess the impact of these variants on gene expression variation, we explored a half-diallel panel composed of 323 hybrids originated from pairwise crosses of 26 natural Saccharomyces cerevisiae isolates. Using short- and long-read sequencing strategies, we established an exhaustive catalog of single nucleotide polymorphisms (SNPs) and SVs for this panel. Combining this dataset with the transcriptomes of all hybrids, we comprehensively mapped SNPs and SVs associated with gene expression variation. While SVs impact gene expression variation, SNPs exhibit a higher effect size with an overrepresentation of low-frequency variants compared to common ones. These results reinforce the importance of dissecting the heritability of complex traits with a comprehensive catalog of genetic variants at the population level.

Isolation and identification of yeasts from local fruits in Thua Thien Hue province, Vietnam

In this study, a total of 30 yeast isolates were recovered from local fruits in Thua Thien Hue province. Genetic characterization based on the ITS sequences identified isolates belonging to 3 species including Saccharomyces cerevisiae, Lachancea fermentati, and Clavispora fructus, with high sequence homology (over 99%) compared to published sequences in the GenBank. All identified S. cerevisiae isolates could grow well at 30°C and ferment several sugar including fructose, galactose, sucrose, mannose, maltose, and raffinose with different performances, but were inhibited at temperature higher than 35°C. The strains also grew well in the medium containing 5% ethanol (v/v) and 200 g/L glucose, but their growth ability was decreased gradually with an increase in ethanol and glucose concentrations. Interestingly, D14 strain was able to grow in the medium supplemented with 12% of ethanol, and 500 g/L of glucose at 45°C, while D7 strain could utilize both mannitol and glycerol at a low level. Our results also indicated that some strains have relatively high sedimentation efficiency, which are favorable conditions for beer fermentation and biomass recovery. The isolated yeast strains with good tolerance properties may provide a potential source of valuable raw materials for applications in beverage production and food processing.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

Novel insights into the effects of 5-hydroxymethfurural on genomic instability and phenotypic evolution using a yeast model.

5-Hydroxymethfurural (5-HMF) is naturally found in a variety of foods and beverages and represents a main inhibitor in the lignocellulosic hydrolysates used for fermentation. This study investigated the impact of 5-HMF on the genomic stability and phenotypic plasticity of the yeast Saccharomyces cerevisiae. Using next-generation sequencing technology, we examined the genomic alterations of diploid S. cerevisiae isolates that were subcultured on a medium containing 1.2 g/L 5-HMF. We found that in 5-HMF-treated cells, the rates of chromosome aneuploidy, large deletions/duplications, and loss of heterozygosity were elevated compared with that in untreated cells. 5-HMF exposure had a mild impact on the rate of point mutations but altered the mutation spectrum. Contrary to what was observed in untreated cells, more monosomy than trisomy occurred in 5-HMF-treated cells. The aneuploidy mutant with monosomic chromosome IX was more resistant to 5-HMF than the diploid parent strain because of the enhanced activity of alcohol dehydrogenase. Finally, we found that overexpression of ADH6 and ZWF1 effectively stabilized the yeast genome under 5-HMF stress. Our findings not only elucidated the global effect of 5-HMF on the genomic integrity of yeast but also provided novel insights into how chromosomal instability drives the environmental adaptability of eukaryotic cells.IMPORTANCESingle-cell microorganisms are exposed to a range of stressors in both natural and industrial settings. This study investigated the effects of 5-hydroxymethfurural (5-HMF), a major inhibitor found in baked foods and lignocellulosic hydrolysates, on the chromosomal instability of yeast. We examined the mechanisms leading to the distinct patterns of 5-HMF-induced genomic alterations and discovered that chromosomal loss, typically viewed as detrimental to cell growth under most conditions, can contribute to yeast tolerance to 5-HMF. Our results increased the understanding of how specific stressors stimulate genomic plasticity and environmental adaptation in yeast.

Non-additive genetic components contribute significantly to population-wide gene expression variation

Gene expression variation, an essential step between genotype and phenotype, is collectively controlled by local (cis) and distant (trans) regulatory changes. Nevertheless, how these regulatory elements differentially influence gene expression variation remains unclear. Here, we bridge this gap by analyzing the transcriptomes of a large diallel panel consisting of 323 unique hybrids originating from genetically divergent Saccharomyces cerevisiae isolates. Our analysis across 5,087 transcript abundance traits showed that non-additive components account for 36% of the gene expression variance on average. By comparing allele-specific read counts in parent-hybrid trios, we found that trans-regulatory changes underlie the majority of gene expression variation in the population. Remarkably, most cis-regulatory variations are also exaggerated or attenuated by additional trans effects. Overall, we showed that the transcriptome is globally buffered at the genetic level mainly due to trans-regulatory variation in the population.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4–4.5 g/L, representing 35–40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production.Key points• Process-related fermentation stresses influence heterologous protein production.• Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP.• CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Technological and acid stress performance of yeast isolates from industrial sourdough

Yeast populations in sourdough have been poorly characterized from baking-relevant attributes as leavening activity in a stressful environment. In particular, the presence of extracellular acids represents an important challenge, which compromises the growth and fermentative metabolism of microbial starters. Here, we have analyzed the fungal diversity of six industrial sourdoughs identifying Saccharomyces cerevisiae and Kazachstania humilis as the predominant species. The volatile organic compounds (VOC) profile revealed differences between sourdough samples which can be attributed, at least in part, to their microbial composition and ingredients used in each one. In particular, K. humilis appeared to contribute to the presence octan-1-ol, 2-hepten-1-ol, 2-octen-1-ol, nonan-1-ol and 2-pentylfuran in the sourdough. A total of 45 yeast isolates were then analyzed in terms of CO2 production and tolerance to NaCl, ethanol, oxidative-, cold- and heat-stress, which allowed the selection of 5 S. cerevisiae isolates and 2 from both K. humilis and Torulaspora delbrueckii for further characterization. Overall, K. humilis isolates showed a higher total CO2 production and gassing rate than a commercial yeast strain used as a control. Hence, the isolated strains are potential candidates for the development of new sourdough starters and the basis for a further selection of variants with superior performance.

Natural variation in codon bias and mRNA folding strength interact synergistically to modify protein expression in Saccharomyces cerevisiae.

Codon bias and mRNA folding strength (mF) are hypothesized molecular mechanisms by which polymorphisms in genes modify protein expression. Natural patterns of codon bias and mF across genes as well as effects of altering codon bias and mF suggest the influence of these two mechanisms may vary depending on the specific location of polymorphisms within a transcript. Despite the central role codon bias and mF may play in natural trait variation within populations, systematic studies of how polymorphic codon bias and mF relate to protein expression variation are lacking. To address this need, we analyzed genomic, transcriptomic, and proteomic data for 22 Saccharomyces cerevisiae isolates, estimated protein accumulation for each allele of 1620 genes as the log of protein molecules per RNA molecule (logPPR), and built linear mixed effects models associating allelic variation in codon bias and mF with allelic variation in logPPR. We found codon bias and mF interact synergistically in a positive association with logPPR and this interaction explains almost all the effect of codon bias and mF. We examined how the locations of polymorphisms within transcripts influence their effects and found that codon bias primarily acts through polymorphisms in domain encoding and 3' coding sequences while mF acts most significantly through coding sequences with weaker effects from UTRs. Our results present the most comprehensive characterization to date of how polymorphisms in transcripts influence protein expression.

Screening and Optimization Consortium Technique of the Bacillus megatherium and Saccharomyces cerevisiae Microbial Consortium for Ethanol Detection

Determination of alcohol content is very important in the food and beverage industry. Biosensor is an alternative to measuring ethanol content. Alcohol biosensors with a single microbe still have a narrow measurement area at ethanol concentrations, so a microbial consortium is needed to widen the range of measured ethanol concentrations. Therefore, it is necessary to screen the microbes from Bacillus sp and S. cerevisiae which have the potential to produce alcohol dehydrogenase (ADH) enzymes and optimize the consortium technique that can provide the best response to oxidation currents. In the yeast microbial screening of 14 S. cerevisiae isolates and 5 Bacillus sp isolates, it can be concluded that for the 14 yeast S. cerevisiae isolates that have the potential to produce ADH enzymes, there is one isolate with the SCRF code. For the 5 bacterial isolates of Bacillus sp that have the potential to produce ADH enzymes, all Bacillus isolates with the code Bacillus megatherium 29/9/14, Bacillus megatherium 23/6/22, Bacillus 6, Bacillus 53, and Bacillus 55. Based on the oxidation current data, Bacillus megatherium 23 /6/22 produces the highest current compared to other Bacillus isolates. The consortium technique that provides the highest current is the method of mixing 1:1 (µL) microbial suspension in an Eppendorf container. Optimization of the consortium's biofilms using the Response Surface Method was produced at 10 days of age, pH 7.5, and 75 µL of microbial suspension dripping volume.

Populations of Saccharomyces cerevisiae in Vineyards: Biodiversity and Persistence Associated with Terroir

The origin terroir provides distinctive characteristics for wines, in relation to soil, climate, oenological practices, etc. Hence, the characterization of each wine region by multiple aspects would allow differentiation of its wines. Several approaches at different scales have studied terroir microbiological fingerprints: from global microbiome analysis up to intraspecific Saccharomyces biodiversity. Mature grapes are the primary source of yeasts, and S. cerevisiae is a key wine fermentative species. Malbec is the emblematic Argentinean variety and is mainly cultivated in the “Zona Alta del Rio Mendoza” (ZARM). In this work, the diversity of S. cerevisiae grape populations was studied at three vintages in two Malbec vineyards of the ZARM, to evaluate their annual diversity and behavior in different vintages. Rarefaction of classical ecological indices was applied for a statistically adequate biodiversity analysis. A total of 654 S. cerevisiae isolates were differentiated by Interdelta-PCR. Each yeast grape population showed a unique composition of S. cerevisiae strains; however, a narrow genetic relationship was found in each vineyard. A slight increase in the initial diversity and a stabilization in the diversity of S. cerevisiae populations were confirmed. These results add to the discussion about the contribution of yeasts to the terroir microbiological concept, and its limitations and stability over the time.

Mixed yeast communities contribute to regionally distinct wine attributes

There is evidence that vineyard yeast communities are regionally differentiated, but the extent to which this contributes to wine regional distinctiveness is not yet clear. This study represents the first experimental test of the hypothesis that mixed yeast communities-comprising multiple, region-specific, isolates, and species-contribute to regional wine attributes. Yeast isolates were sourced from uninoculated Pinot Noir fermentations from 17 vineyards across Martinborough, Marlborough, and Central Otago in New Zealand. New methodologies for preparing representative, mixed species inoculum from these significantly differentiated regional yeast communities in a controlled, replicable manner were developed and used to inoculate Pinot Noir ferments. A total of 28 yeast-derived aroma compounds were measured in the resulting wines via headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Yeast community region of origin had a significant impact on wine aroma, explaining ∼10% of the observed variation, which is in line with previous reports of the effects of region-specific Saccharomyces cerevisiae isolates on Sauvignon Blanc ferments. This study shows that regionally distinct, mixed yeast communities can modulate wine aroma compounds in a regionally distinct manner and are in line with the hypothesis that there is a microbial component to regional distinctiveness, or terroir, for New Zealand Pinot Noir.