Bioprospecting Australian microbial genetic diversity
An important aspect of terroir, particularly where spontaneous fermentations are performed, is very likely to be related to differences in wine microbiota. Focused microbiological research has shown that both vineyards and spontaneous fermentations contain diverse mixtures of microbial species (often with species being represented by multiple strains). However, the inability to transition this traditional methodology to efficiently and accurately assess the large numbers of samples required to tackle such a complex problem, has so far limited subsequent insights into this important question. This lack of information is therefore a major impediment to the exploitation of native microbial germplasm and spontaneous fermentation, by the Australian wine industry.
However, recent advances in metagenomics (genomic sequencing of mixed microbial communities), can address these issues by providing detailed identification of the species, and their proportions which comprise complex microbial mixtures, in a high throughput manner. In addition, by adopting and developing new genomic approaches, such as single cell high throughput sequencing, the genetic make-up of individual strains within these mixtures can also be obtained, to provide direct links between novel genetic and phenotypic characteristics. This type of metagenomic analysis has been initiated and refined for studies of wine fermentation at the AWRI, and now provides the technical platform to answer important questions regarding Australian wine microbial terroir.
This project builds on an existing project where the contributions of various species of fungi, yeast and bacteria are being elucidated in vineyard-to-wine time course experiments, using samples sourced from the spectrum of Australian wine regions, highlighting both the temporal and geographic dynamics of the microbial populations. Once these regional differences are identified, key microbes will be isolated in pure culture to assess their contributions to the unique terroir or wine style(s) of the region. In addition to regional wine isolates sourced from the metagenomics project, wild yeast strains will be sought from non-winemaking areas of Australia, as potential reservoirs of new and desirable winemaking characteristics, and uniquely Australian germplasm.
The thousands of strains that are isolated by this project, in addition to those already present in the AWRI Wine Microorganism Culture Collection, will be then assessed by high-throughput screening for desirable winemaking properties such as the production of key aroma compounds, the production of enzymes, and the ability to produce lower alcohol concentrations.
In addition, common winemaker interventions in wild fermentations will be explored by subjecting fermentations to various external factors (e.g. sulfite, oxygen, temperature, and/or high solids juice), to determine if it is possible to rationally influence or shape the performance or style of wild fermentations.
Strains that exhibit desirable phenotypes will progress to laboratory-scale fermentation and potentially to winery-scale and external trials, depending on their behaviour. These characterised strains will therefore provide a key resource for wineries to enhance the expression of regional terroir through inoculation of existing microbes either in the vineyard or within individual ferments.
Winemaking interventions to influence the microbial composition of wild ferments
Of the fermentation conditions that are readily modulated by winemakers, the addition of SO2 represents the most broadly available intervention practice. Previous microbiological studies have shown that species (and strains) of the major wine yeasts can respond differently to the application of this antimicrobial agent. Typically, commercial strains of Saccharomyces cerevisiae display high tolerance to SO2, while ‘wild’ yeasts display lower tolerances and are therefore thought to be broadly selected against through the application of moderate amounts of SO2 prior to the start of fermentation.
Winemaking trials were performed across the 2018, 2019 and 2020 vintages, focusing on the ability of SO2 additions to influence the microbial and chemical composition of wild fermentations. The addition of as little as 35 mg/L of SO2 prior to the initiation of fermentation was shown to significantly, and reproducibly, alter the non-Saccharomyces yeast composition of the ferments. This was shown to be accompanied by changes in the final chemical composition of the wines, with significantly higher levels of acetate esters being observed in the wines made from SO2-treated juice (Figure 1).
The ability to use SO2 to predictably modulate the yeast community structure of a wild ferment, and the resulting chemical composition of the final wine, represents an important tool for winemakers to begin to be able to influence the sensory profile of wines made from wild fermentations.
High-throughput assays have been used to screen a collection of more than 2,000 non-Saccharomyces yeast strains in order to identify isolates with winemaking potential. These screens include the ability for the isolates to grow on high-sugar substrates and in high-ethanol environments as well as the ability to produce several different enzyme activities that have been associated with the release of aroma compounds from non-volatile precursors in grape juice.
Genome sequencing of several wine isolates of the genus Aureobasidium has uncovered three potentially new species within this genus of yeast-like fungi that forms part of the natural flora of grapevines, and which may play an important role early in fermentation.
Anthony Borneman, Cristian Varela