Hanseniaspora uvarum is consistently observed as the dominant non-Saccharomyces species in grape juice leading to spontaneous fermentations and is often considered a detrimental factor due to the increased volatile acidity associated with its abundance. This study investigated how fermentation temperature affects the competitive interaction between H. uvarum and Saccharomyces cerevisiae, and how these shifts influence microbial community structure during spontaneous grape juice fermentations. Defined single- and co-inoculated fermentations were conducted across a range of temperatures (17, 23, and 25 °C), and cell abundance was quantified by flow cytometry. In parallel, spontaneous fermentations were monitored using ITS metabarcoding to assess temperature-driven changes in fungal community composition. At 17 °C, H. uvarum dominated early fermentation due to its faster growth rate, resulting in slower sugar consumption and prolonged fermentations. Increasing fermentation temperature enhanced the early growth of S. cerevisiae. Consistent patterns were observed in spontaneous fermentations, where higher temperatures increased the relative abundance of S. cerevisiae and shortened fermentation time. These results demonstrate that fermentation temperature is a major driver of yeast competition and suggest that moderate increases in fermentation temperature may help limit excessive H. uvarum proliferation during spontaneous fermentations.

Hanseniaspora uvarum is consistently observed as the dominant non-Saccharomyces species in spontaneous grape juice fermentations. However, the physiological mechanisms and physicochemical variables influencing the prevalence of H. uvarum over other non-Saccharomyces species remain unclear. We tested the factors contributing to H. uvarum dominance by inoculating a chemically diverse set of grape juices with a mock community whose composition was based on a previously published comprehensive microbial survey of commercial spontaneous fermentations. The diverse composition of these grape juices appeared to have minimal impact on the overall microbial dynamics of fermentation, with H. uvarum consistently emerging as the dominant non-Saccharomyces species in nearly all conditions tested. Flow cytometry analysis confirmed that H. uvarum has a faster growth rate than Saccharomyces cerevisiae and several other Hanseniaspora species. Moreover, its growth was not affected by the presence of S. cerevisiae. H. uvarum negatively affected the growth of S. cerevisiae, with significant implications for fermentation performance and sugar consumption. Our study suggests that the fast growth rate of H. uvarum enables it to dominate the grape juice environment quickly during early fermentation stages. This physiological advantage may be critical to the outcome of spontaneous fermentations, as evidenced by its direct impact on S. cerevisiae and fermentation performance.

The genus Hanseniaspora includes apiculate yeasts commonly found in fruit- and fermentation-associated environments. Their genetic diversity and evolutionary adaptations remain largely unexplored despite their ecological and oenological significance. This study investigated the phylogenetic relationships, genome structure, selection patterns, and phenotypic diversity of Hanseniaspora species isolated primarily from Australian wine environments, focusing on Hanseniaspora uvarum, the most abundant non-Saccharomyces yeast in wine fermentation. A total of 151 isolates were sequenced, including long-read genomes for representatives of the main phylogenetic clades. Comparative genomics revealed ancestral chromosomal rearrangements between the slow-evolving lineage (SEL) and fast-evolving lineage (FEL) that could have contributed to their evolutionary split, as well as significant loss of genes associated with mRNA splicing, chromatid segregation and signal recognition particle protein targeting in the FEL. Pangenome analysis within H. uvarum identified extensive copy number variation, particularly in genes related to xenobiotic tolerance and nutrient transport. Investigation into the selective landscape following the FEL/SEL divergence identified diversifying selection in 229 genes in the FEL, with significant enrichment in genes within the lysine biosynthetic pathway. Furthermore, phenotypic screening of 116 isolates revealed substantial intraspecific diversity, with specific species exhibiting enhanced ethanol, osmotic, copper, SO₂, and cold tolerance.

Extensive research has been dedicated to elucidating the role of various nitrogen sources, nitrogen concentrations and the timing of addition when modulating grape must fermentation using yeast. The wine industry invests substantial resources in both the vineyard and the winery to provide adequate nitrogen concentrations for fermentation. This approach ensures optimal yeast performance during fermentation and minimises the risk of negative sensory attributes associated with poor ferment nutrition. In addition to wine, the winemaking process produces a substantial quantity of nutrient-rich biomass, a poorly explored resource that, if appropriately recycled, could be used to support the nutrient requirements of other winery fermentations. This study explored the feasibility of using processed yeast lees generated during alcoholic fermentation as a nutrient supplement in subsequent fermentations. Three lees treatment options were assessed: accelerated autolysis, enzymatic lysis and mechanical lysis. The ability of these treatments to achieve complete lysis of yeast cells and release amino acids and trace elements is reported. The addition of processed lysates into grape juice was shown to improve fermentation timeframes and influence the production of yeast-derived fermentation volatile compounds in a dose-dependent manner. This study demonstrates that recycling spent lees from winery waste is feasible and provides some strategies for extracting nutrients from winery waste.

Background and Aims. An OIV resolution provides guidelines on using glutathione as a prefermentation additive when the amount of yeast assimilable nitrogen (YAN) of a juice or must is adequate, to avoid the metabolism of glutathione by the yeast. The effect of YAN concentration on glutathione metabolism by yeast had not been determined. This study explored whether nitrogen management could be used to control glutathione consumption during fermentation. Methods and Results. An HPLC-UV method was developed to quantify reduced L-glutathione (GSH) and oxidised glutathione (GSSG) and used to monitor yeast GSH metabolism during alcoholic fermentation with two yeast strains (AWRI 1688 and AWRI 2861). The addition of GSH had no impact on the fermentation rate of the chemically defined medium, even in a limited YAN environment; however, a decrease in glutathione concentration occurred regardless of YAN concentration. The effect of GSH on volatile sulfur compound formation was yeast strain-dependent. Conclusions. Increasing the YAN status of a chemically defined medium led to a decrease in GSH consumption during fermentation, but the loss of GSH could not be prevented entirely, even with a low initial GSH concentration and high initial YAN. Significance of the Study. In the presence of higher concentrations of GSH during fermentation, there is a risk of forming undesirable fermentative sulfur compounds that are not mitigated through nitrogen supplementation. Thus, it seems unlikely that an argument could be made for the inclusion of GSH in relevant food standards codes as a wine additive especially if a lack of GSH metabolism was a criterion.

Stuck or sluggish malolactic fermentation (MLF) can be problematic in stressful wine conditions, particularly white and sparkling base musts/wines. In these cases, knowledge of yeast-bacteria strain compatibility and the amount of sulfur dioxide (SO2) a yeast strain produces are important considerations for successful MLF. Here, laboratory- and pilot-scale co-fermentations in Chardonnay were used to investigate the effect of yeast-derived SO2 on Oenococcus oeni survival. Although yeast-derived SO2 is generally inhibitory, we show that SO2 production (to approximately 65 mg/L) can be uncoupled from O. oeni survival in the early stages of co-fermentation. Bacterial survival in the presence of specific SO2-producing yeast strains was correlated with the early, transient formation of high acetaldehyde concentrations. Oenococcus oeni survival coincided with molecular SO2 concentrations remaining below an extremely low threshold of inhibition, which exponentially increased from approximately 3–6 µg/L in the first three days of co-fermentation. Strain-dependent sensitivity of O. oeni to bound SO2 remains a possibility, although the extent and mechanism of such inhibition by the SO2 adduct during co-fermentation remain unclear. The choice of co-inoculation yeast strain also influenced wine diacetyl concentration, which was only detected in wines co-inoculated with high SO2-producing S. cerevisiae strains. The wines with high diacetyl concentrations were found to be distinct by a sensory panel, with comparatively high citation frequency for a buttery sensory attribute. Both the SO2- and acetaldehyde production capacity of yeasts are, therefore, seen as meaningful co-inoculation selection criteria. The range of yeast strains suitable for MLF induction by co-inoculation could be widened to include SO2-producing strains that transiently produce an early, high concentration of acetaldehyde. The effects of low, equilibrium concentrations of molecular SO2 should also be considered in conjunction with total SO2 as a measure of SO2 toxicity towards O. oeni following co-inoculation.

Copper tolerance and SO 2 tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae . The genetic bases of these traits are the allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between SO 2 and copper tolerance in S . cerevisiae wine yeasts. Here we probe the relationship between SO 2 and copper tolerance and show that an increase in CUP1 copy number does not always impart copper tolerance in S . cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1 . Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production and provided evidence that SSU1 over-expression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16 , genes upstream of H 2 S production in the sulfate assimilation pathway increased the production of SO 2 and H 2 S but did not improve copper sensitivity in an SSU1 over-expressing background. We conclude that copper and SO 2 tolerance are conditional traits in S . cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary driver for the extreme amplification of CUP1 observed in some yeasts.

During the alcoholic fermentation of grape sugars, wine yeast produces a range of secondary metabolites that play a critical role in the aroma profile of wines. One of the most impactful yeast-modified compound families, particularly in white wines, are the ‘fruity’ polyfunctional thiols, which include 3-mercaptohexan-1-ol (3-MH) and 4-mercapto-4-methylpentan-2-one (4-MMP). While the formation and stylistic contribution of these thiols have been extensively researched in white wines, little is known about the conditions leading to their formation in red wines. In this study, we explored the ability of yeast strains to modulate the release of these aroma compounds during the fermentation of two red musts. In laboratory-scale Pinot Noir fermentations, the formation of 3-MH strongly correlated with yeast β-lyase activity, particularly with the presence of certain genotypes of the flavour-releasing gene IRC7. Subsequent production of Grenache wine at the pilot scale, with detailed compositional and sensory analysis, was undertaken to confirm laboratory-scale observations. A commercial wine strain used for expressing ‘fruity’ thiols in Sauvignon Blanc was shown to produce wines that exhibited more intense red fruit aromas. These results reveal an opportunity for winemakers to shape red wine aroma and flavour by using yeasts that might typically be considered for white wine production.

To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive hetero-fermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

Background and Aims Aeration during fermentation of red wines has the potential to enhance positive red fruit attributes and suppress less desirable reductive characteristics. The implementation of aeration in commercial winemaking, however, is impeded by the lack of a clear understanding of both the major benefits of its use and the nature of the aeration regimes required to modify the finished product. This work aimed to evaluate the impact of different modes of aeration, varying in their timing, duration and intensity, on fermentation duration, chemical composition and sensory properties of Shiraz wine. Methods and Results Forty-eight pilot-scale fermentations (450 kg) were treated with different aeration modalities and compared to non-aerated fermentations across four vintages. This work demonstrated the reproducible effects of aeration, resulting in an enhancement of fruit-related attributes, decreases in colour intensity and a lowering of astringent mouthfeel and bitterness. The development of attributes describing possible oxidation or undesirable volatility were not observed in this work. These effects on wine sensory attributes were correlated with an increase in the concentration of relevant short and branched-chain esters and acetates, and a decrease in the concentration of phenolic substances. No enhancement of fermentation performance was observed in any of the trials. Overall, this work revealed the robustness of red wine fermentations to aeration. Conclusions This work provides strong evidence that aeration does not alter the duration of either alcoholic or malolactic fermentation of red musts. Although there was no impact on fermentation performance, aeration did result in the production of wines with increased red fruit characters, and decreased astringency and reductive off-odours. Significance of the Study This work demonstrates aeration to be a useful oenological tool for the production of red wine and indicates that stylistic impact, rather than fermentation performance, is likely to be more pertinent to the adoption of red wine aeration.

Uninoculated wines are regarded as having improved mouthfeel and texture and more complex flavor profiles when compared to wines inoculated with commercial S. cerevisiae strains. Uninoculated fermentation involves a complex microbial succession of yeasts and bacteria during fermentation. Microbial population dynamics are affected by several factors that can ultimately determine if a particular species or strain contributes to wine aroma and flavor. In this work, we have studied the effect of aeration, a common winemaking practice, on the yeast microbiota during uninoculated Chardonnay wine fermentation. The timing of aeration and then aeration intensity were evaluated across two successive vintages. While the timing of aeration significantly impacted fermentation efficiency across oxygen treatments, different levels of aeration intensity only differed when compared to the non-aerated control ferments. Air addition increased the viable cell population size of yeast from the genera Hanseniaspora, Lachancea, Metschnikowia and Torulaspora in both vintages. While in 2019, a high relative abundance was found for Hanseniaspora species in aerated ferments, in 2020, T. delbrueckii was visibly more abundant than other species in response to aeration. Accompanying the observed differences in yeast community structure, the chemical profile of the finished wines was also different across the various aeration treatments. However, excessive aeration resulted in elevated concentrations of ethyl acetate and acetic acid, which would likely have a detrimental effect on wine quality. This work demonstrates the role of aeration in shaping yeast population dynamics and modulating a volatile profile in uninoculated wines, and highlights the need for careful air addition to avoid a negative sensory impact on wine flavor and aroma.

The use of non-Saccharomyces yeast species generally involves sequential or co-inoculation of a Saccharomyces cerevisiae strain to complete fermentation. While most studies have focused on characterising the impact that S. cerevisiae has on the growth and metabolic activity of these non-Saccharomyces species, microbial interactions work reciprocally. Antagonism or competition of non-Saccharomyces species against S. cerevisiae has been shown to impact subsequent fermentation performance. To date, it remains unclear whether these negative interactions are strain specific. Hence, characterisation of strain-specific responses to co-inoculation would enable the identification of specific S. cerevisiae strain/non-Saccharomyces combinations that minimise the negative impacts of sequential fermentation on fermentation performance. The competitive fitness response of 93 S. cerevisiae strains to several non-Saccharomyces species was simultaneously investigated using a barcoded library to address this knowledge gap. Strain-specific fitness differences were observed across non-Saccharomyces treatments. Results obtained from experiments using selected S. cerevisiae strains sequentially inoculated after Metschnikowia pulcherrima and Torulaspora delbrueckii were consistent with the competitive barcoded library observations. The results presented in this study indicate that strain selection will influence fermentation performance when using non-Saccharomyces species, therefore, appropriate strain/yeast combinations are required to optimise fermentation.

Sparkling wine made by the traditional method (Méthode Traditionelle) develops a distinct and desirable flavour and aroma profile attributed to proteolytic processes during prolonged ageing on lees. Microwave, ultrasound and addition of β-glucanase enzymes were applied to accelerate the disruption of Saccharomyces cerevisiae, and added to the tirage solution for secondary fermentation in traditional sparkling winemaking. Scanning electron microscopy and flow cytometry analyses were used to observe and describe yeast whole-cell anatomy, and cell integrity and structure via propidium iodide (PI) permeability after 6-, 12- and 18-months post-tirage. Treatments applied produced features on lees that were distinct from that of the untreated control yeast. Whilst control yeast displayed budding cells (growth features) with smooth, cavitated and flat external cell appearances; microwave treated yeast cells exhibited modifications like ‘doughnut’ shapes immediately after treatment (time 0). Similar ‘doughnut’-shaped and ‘pitted/porous’ cell features were observed on progressively older lees from the control. Flow cytometry was used to discriminate yeast populations; features consistent with cell disruption were observed in the microwave, ultrasound and enzyme treatments, as evidenced by up to 4-fold increase in PI signal in the microwave treatment. Forward and side scatter signals reflected changes in size and structure of yeast cells, in all treatments applied. When flow cytometry was interpreted alongside the scanning electron microscopy images, bimodal populations of yeast cells with low and high PI intensities were revealed and distinctive ‘doughnut’-shaped cell features observed in association with the microwave treatment only at tirage, that were not observed until 12 months wine ageing in older lees from the control. This work offers both a rapid approach to visualise alterations to yeast cell surfaces and a better understanding of the mechanisms of yeast lysis. Microwave, ultrasound or β-glucanase enzymes are tools that could potentially initiate the release of yeast cell compounds into wine. Further investigation into the impact of such treatments on the flavour and aroma profiles of the wines through sensory evaluation is warranted.

The higher alcohols 2-phenylethanol, tryptophol, and tyrosol are a group of yeast-derived compounds that have been shown to affect the aroma and flavour of fermented beverages. Five variants of the industrial wine strain AWRI796, previously isolated due to their elevated production of the 'rose-like aroma' compound 2-phenylethanol, were characterised during pilot-scale fermentation of a Chardonnay juice. We show that these variants not only increase the concentration of 2-phenylethanol but also modulate the formation of the higher alcohols tryptophol, tyrosol, and methionol, as well as other volatile sulfur compounds derived from methionine, highlighting the connections between yeast nitrogen and sulfur metabolism during fermentation. We also investigate the development of these compounds during wine storage, focusing on the sulfonation of tryptophol. Finally, the sensory properties of wines produced using these strains were quantified at two time points, unravelling differences produced by biologically modulating higher alcohols and the dynamic changes in wine flavour over aging.

Premium sparkling wine produced by the traditional method (analogous to the French méthode champenoise) is characterised by the development of aged wine character as a result of a second fermentation in the bottle with lees contact and lengthy ageing. Treatments (microwave, ultrasound, or β-glucanase enzymes) were applied to disrupt the cell wall of Saccharomyces cerevisiae and added to the tirage liquor for the second fermentation of Chardonnay-Pinot Noir base wine cuvée and compared to a control, to assess effects on the release of phenolics, proteins, amino acids, and lipids at 6, 12 and 18 months post-tirage. General responses to wine ageing included a 60% increase in the total phenolic content of older sparkling wines relative to younger wines and an increase in protein concentration from 6 to 12 months bottle age. Microwave and β-glucanase enzyme treatments of yeast during tirage preparation were associated with a 10% increase in total free amino acid concentration and a 10% increase in proline concentration at 18 months bottle age, compared to control and ultrasound treatment. Furthermore, microwave treatment was associated with elevated asparagine content in wine at 18 months bottle age, relative to the control and the other wines. The β-glucanase enzyme and ultrasound treatments were associated with significant accumulation of total lipids, which were driven by 2-fold increases in the phospholipid and monoacylglycerol components in wine at 18 months bottle age and, furthermore, the microwave treatment was associated with elevated triacylglycerol at 18 months bottle age. This study demonstrates that the use of yeast treatments at the tirage stage of sparkling wine production presents an opportunity to manipulate wine composition.

Background and Aims The Chardonnay clone Gingin was imported into Western Australia in 1957 and has become one of Australia's oldest recognised Chardonnay clones. While its phenotypic similarity to other Chardonnay clones has been noted, its origins and relatedness to these clones remains a mystery. This study builds on previous Chardonnay clonal marker research to uncover the origins of Gingin. Methods and Results Samples of Gingin and clones suspected to be related to Gingin were sequenced and analysed in a marker discovery pipeline along with previously sequenced Chardonnay clones. Sequencing data for previously uncharacterised samples were also included. Clonal markers were identified for all samples and a new phylogeny for all sequenced Chardonnay clones was built which revealed that Gingin is related to other clones. Conclusions The results are consistent with Gingin being a selection of an old University of California, Davis/Foundation Plant Services clone called Chardonnay-1, from which the clone OF Chard was derived. It is also related to the clone Mendoza, but the origin of this clone is still unclear. Significance of the Study This study demonstrates the potential of clonal genetic markers for the identification of grapevine clones, and in helping to unravel their origin and to establish their heritage; in this example shedding light on a 60-year-old mystery and opening the door for authenticity testing of these important clones.

Aureobasidium pullulans has been observed as one of the most abundant species in freshly pressed grape juice. Despite this, little is known about the consequences for the wine-making process associated with the presence and proliferation of this fungus, including its interaction with other ferment-derived microorganisms and impact on the composition of the resulting wine. In this study, the physiology of abundant A. pullulans grape juice isolates was investigated through lab scale fermentation trials, demonstrating the ability of this species to survive in grape juice while producing polysaccharides, polymers of malic acid (poly β-malic acid) and enzymes with pectinase, β - glucosidase and tannase activity. A possible antagonistic effect against yeast through competition for metals including Fe and Zn was also observed. Overall, the data suggests this abundant species could have important implications for wine production and quality.

Uninoculated wine fermentations are conducted by a consortium of wine yeast and bacteria that establish themselves either from the grape surface or from the winery environment. Of the additives that are commonly used by winemakers, sulphur dioxide (SO2) represents the main antimicrobial preservative and its use can have drastic effects on the microbial composition of the fermentation. To investigate the effect of SO2 on the resident yeast community of uninoculated ferments, Chardonnay grape juice from 2018 and 2019 was treated with a variety of SO2 concentrations ranging up to 100 mg/L and was then allowed to undergo fermentation, with the yeast community structure being assessed via high-throughput meta-barcoding (phylotyping). While the addition of SO2 was shown to select against the presence of many species of non-Saccharomyces yeasts, there was a clear and increasing selection for the species Hanseniaspora osmophila as concentrations of SO2 rose above 40 mg/L in fermentations from both vintages. Chemical analysis of the wines resulting from these treatments showed significant increases in acetate esters, and specifically the desirable aroma compound 2-phenylethyl acetate, that accompanied the increase in abundance of H. osmophila. The ability to modulate the yeast community structure of an uninoculated ferment and the resulting chemical composition of the final wine, as demonstrated in this study, represents an important tool for winemakers to begin to be able to influence the organoleptic profile of uninoculated wines.

When a wine yeast is inoculated into grape juice the potential variation in juice composition that confronts it is huge. Assessing the performance characteristics of the many commercially available wine yeasts in the many possible grape juice compositions is a daunting task. To this end we have developed a barcoded Saccharomyces cerevisiae wine yeast collection to facilitate the task of performance assessment that will contribute to a broader understanding of genotype-phenotype relations. Barcode sequencing of mixed populations is used to monitor strain abundance in different grape juices and grape juice-like environments. Choice of DNA extraction method is shown to affect strain-specific barcode count in this highly related set of S. cerevisiae strains; however, the analytical approach is shown to be robust toward strain dependent variation in DNA extraction efficiency. Of the 38 unique compositional variables assessed, resistance to copper and SO2 are found to be dominant discriminatory factors in wine yeast performance. Finally, a comparison of competitive fitness profile with performance in single inoculum fermentations reveal strain dependent correspondence of yeast performance using these two different approaches.