Kombucha fermentation is initiated by transferring a solid-phase cellulosic pellicle into sweetened tea and allowing the microbes that it contains to initiate the fermentation. This pellicle, commonly referred to as a symbiotic culture of bacteria and yeast (SCOBY), floats to the surface of the fermenting tea and represents an interphase environment, where embedded microbes gain access to oxygen as well as nutrients in the tea. To date, various yeast and bacteria have been reported to exist within the SCOBY, with little consensus as to which species are essential and which are incidental to Kombucha production. In this study, we used high-throughput sequencing approaches to evaluate spatial homogeneity within a single commercial SCOBY and taxonomic diversity across a large number (n = 103) of SCOBY used by Kombucha brewers, predominantly in North America. Our results show that the most prevalent and abundant SCOBY taxa were the yeast genus Brettanomyces and the bacterial genus Komagataeibacter, through careful sampling of upper and lower SCOBY layers. This sampling procedure is critical to avoid over-representation of lactic acid bacteria. K-means clustering was used on metabarcoding data of all 103 SCOBY, delineating four SCOBY archetypes based upon differences in their microbial community structures. Fungal genera Zygosaccharomyces, Lachancea and Starmerella were identified as the major compensatory taxa for SCOBY with lower relative abundance of Brettanomyces. Interestingly, while Lactobacillacae was the major compensatory taxa where Komagataeibacter abundance was lower, phylogenic heat-tree analysis infers a possible antagonistic relationship between Starmerella and the acetic acid bacterium. Our results provide the basis for further investigation of how SCOBY archetype affects Kombucha fermentation, and fundamental studies of microbial community assembly in an interphase environment.

To gain knowledge on the role of Saccharomyces cerevisiae yeast strains (and their hybrids) on wine sensory properties, 10 commercially available yeast strains were selected on the basis of their widespread usage and/or novel properties and used to produce Shiraz wines. Significant differences were evident post-alcoholic fermentation and after 24 months of ageing with regards to the number of wine compositional variables, in particular the concentration of tannin and polysaccharide. Strain L2323 is known for its pectinolytic activity and yielded the highest concentration of both yeast- and grape-derived polysaccharides. Wines made with the mannoprotein-producing strain Uvaferm HPS (high levels of polysaccharides) did not have elevated concentrations of yeast-derived polysaccharides, despite this observation being made for corresponding model fermentations, suggesting that mannoprotein production or retention might be limited by the wine matrix. Wine tannin concentration showed a high level of variability between strains, with L2323 having the highest, and AWRI1503 the lowest concentration. Sensory analysis of the wines after 24 months ageing revealed significant differences between the yeast strains, but only the attributes opacity (visual colour) and astringency could be predicted by partial least squares regression using the wine compositional data. Notably, the astringency attribute was associated with higher concentrations of both tannin and polysaccharide, contrary to reports in the literature which suggested that polysaccharide exerts a moderating effect on astringency. The results confirm previous reports demonstrating that the choice of yeast strain represents an opportunity to shape wine style outcomes.

Traditional styles of beer that undergo spontaneous fermentation and/or maturation in oak barrels are becoming increasingly popular amongst craft beer consumers. These products are costly to produce, partly because some barrels develop undesirable flavor profiles and are excluded from the final blend. As a first step towards understanding variation in beer quality that occurs during barrel maturation, next-generation sequencing was used to profile the bacterial microbiome and fungal mycobiome of three beers; an American Coolship Ale (ACA), an aged sour ale, and an Imperial Porter. For the aged sour ale, produced across three years, it was observed that while there were significant differences in key lactic and acetic acid bacterial genera according to beer age, within each production batch there was substantial barrel-induced variation in microbiome profiles. Similarly, for the Imperial Porter significant differences were observed in the relative abundance of Saccharomyces cerevisiae and Brettanomyces bruxellensis across the barrel population at two sampling time points. Overall, the results point towards barrel-induced variation in the rate of “microbial maturation”. Further work to understand the underlying cause of this variation will enable brewers to better predict and monitor their barrel projects to achieve more consistent outcomes.

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.

Kombucha is an acidic non-alcoholic fermented tea beverage that has surged in popularity over the past decade. A key driver of this popularity is the perception that kombucha contains microbes that contribute to gut health, a perception that is reinforced by package claims such as “live” and “probiotic”. We surveyed retail-available non-alcoholic (soft) and alcoholic (hard) kombucha and selected 39 products, of which 74.4% included at least one of these claims. Based upon Nuclear Magnetic Resonance (NMR) evaluation of the chemical composition, approximately one-third of soft kombucha products were re-categorized as “soft-aberrant” on the basis of their alcohol by volume (ABV) exceeding 0.5%. Across all three categories, the majority (82.1%) of products were “live”, containing culturable yeast and/or bacteria. However, there were no significant differences in median maximum colony-forming unit (CFU) counts between these categories, nor was there a difference according to package claims. Interestingly, only 6.3% of soft and 10% of soft-aberrant kombucha products exceeded ${10}^6$ CFU/mL, the threshold that would deliver at least one billion cells in one package. None of the sampled hard kombucha exceeded this threshold. Metabarcoding analyses of microbial communities revealed differences in composition between categories and according to package claims. Notably, the subset of products with “probiotic” claims that listed Bacillus coagulans as an ingredient were enriched in read counts for Bacillus.

The apiculate yeast genus Hanseniaspora has appeared frequently in enological research for more than 100 years, mostly focused upon the species H. uvarum due to its notable capacity to cause spoilage. Recently, there has been increased research into the potential benefits of other Hanseniaspora species, such as H. vineae, in producing more complex wines. Furthermore, large-scale DNA sequencing-based (metabarcoding) vineyard ecology studies have suggested that Hanseniaspora species may not be evenly distributed. To address potential differences across geographical areas in Oregon, we sampled extensively from 12 vineyards within the Willamette Valley American Viticultural Area (AVA), across 2 sub-AVAs (Eola-Amity Hills and Yamhill-Carlton). Metabarcoding was then used to assess the contribution of Hanseniaspora to the grape berry fungal community and the impact of wine processing on diversity. While 6 of the 23 recognized Hanseniaspora species were present on Pinot Noir grapes in the Willamette Valley AVA, differences between vineyards were driven by the abundance of H. uvarum . Significant positive correlations between the amount of H. uvarum present in must and at cold soak, and then cold soak to early ferment were observed. While intuitive, it is worth noting that no prior studies have observed this across such a large number of grape samples from different vineyards. Our results provide clear evidence that the abundance of H. uvarum on grapes may be an important predictor of potential impacts on wine quality, particularly if performing cold soak, which acts as an enrichment step. IMPORTANCE: Hanseniaspora yeasts are frequently found in uninoculated wine fermentations, and depending upon the species present, their contributions to the wine may be positive or negative. We found that in Oregon’s Willamette Valley, the most common species of Hanseniaspora in Pinot Noir vineyards was the known spoilage organism, H. uvarum . This species was one of the strongest contributors to differences in fungal communities between different vineyards and was enriched during typical Pinot Noir processing. These results support Hanseniaspora as an integral and functional component of vineyard “microbial terroir” within Oregon.