Bioprospecting Australian microbial genetic diversity
Abstract
Uninoculated (wild) 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. Wild ferments are considered to provide improved mouthfeel, aroma and flavour complexity compared to inoculated ferments, but at the expense of reliability and reproducibility. This project has applied a combination of genomic and microbiological techniques to investigate Australian wine microbiomes and how non-Saccharomyces yeasts and winemaking interventions can be used shape wine outcomes through altering microbial composition.
Summary
Wild ferments show a far more complex progression of microbial species than inoculated wines and, accordingly, a more complex taste and aroma profile. Differences in these resident microflora between vineyards and wineries are therefore thought to play a key role in defining regional expression of wine characteristics.
To investigate the yeast microbiomes of Australian wild fermentations, a DNA-based yeast profiling framework was established and used to investigate commercial uninoculated Chardonnay and Shiraz wines, with this work later expanded to include Pinot Noir and smaller numbers of ferment samples from other varieties.
Results were obtained from 852 wines from 34 wineries from across 12 Australian winemaking GIs and showed that wines contained a highly variable yeast microbiome that differed across regions, vintages, wineries and vineyards.
While the yeast profiling methodology used for the winery studies provided detailed information on the ferment microbiology, the extended time needed due to centralised processing and complicated sample logistics meant that results were obtained well after the completion of vintage. Other methods were therefore investigated to progress the methodology towards ‘in-field’ testing and a time frame that would allow for ferment intervention based upon analytical data. A method was developed that simultaneously isolated and stored the DNA of mixed microbial samples, which could be used as an alternative to current requirements for cold-chain logistics and DNA extraction. This removed a key roadblock in the transition towards in-field analysis, while also providing a means to streamline logistics for sample storage and transport to centralised locations for processing. It is now conceivable that larger wine laboratories, which can currently handle molecular diagnostics such as qPCR, could perform this technique on-site.
As wild fermentations do not involve the addition of commercial yeast, winemaker intervention is limited, and the effects of interventions on the microbiome of the wine are often poorly understood. Experiments were therefore performed to assess the impact of common and relatively easy to implement interventions - SO2, aeration and temperature changes – on the microbial dynamics and chemical properties of wine.
Given its role as the major antimicrobial used in wine, the effect of SO2 on both the microbiome and wine composition was assessed across four years, moving from laboratory-scale experiments to winery trials. Moderate addition of SO2 prior to the initiation of a wild ferment resulted in a drastic and reproducible change to the yeast species present. This was accompanied by significant changes in the chemical profile of the wines, which could be associated with distinct sensory outcomes.
The effect of aeration on wild fermentation was assessed across three vintages, with initial laboratory-scale experiments also transitioning to winery trials and sensory studies. When provided at the appropriate time, aeration had the potential to aid wild Saccharomyces fermentation efficiency and decrease the time taken for fermentation completion.
Finally, the effect of varying fermentation temperature was investigated in both Chardonnay and Shiraz at laboratory scale. Here, it was shown that increasing temperature allowed for higher cell numbers and faster fermentations, but this came at the expense of wine quality.
These interventions provide a means to alter flavour and aroma outcomes (through the use of SO2) and to affect fermentation efficiency (through the judicious use of aeration). By combining the ability to monitor microbial populations through the yeast profiling techniques with the winemaker interventions that were investigated, the risk of negative wild ferment outcomes can be decreased.
In addition to yeast microbiome profiling, a collection of more than 18,000 wine yeast isolates was established from a range of wild ferment wine samples over a four year period. Species identification indicated that 35 different yeast species were represented in the collection, which provided a foundational germplasm resource that was subsequently used to identify novel non-Saccharomyces isolates for use as commercial starter strains and for wine yeast population genomics studies.
While this project provided information that can be used to lower the perceived risks associated with spontaneous fermentation, it is likely that the majority of wines produced in the Australian market will still make use of inoculation with commercial yeast starter cultures. To address this key market segment, commercial non-Saccharomyces strains can be used to provide novel and/or complex flavour profiles relative to the use of S. cerevisiae alone, while improving fermentation reproducibility and reliability relative to the use of wild fermentation. An iterative screening approach was used to screen a set of almost 1,300 non-Saccharomyces yeast strains collected during this project for desirable winemaking properties. Three candidate strains were progressed through to winery-scale fermentation and full sensory analysis and showed promising characteristics as potential commercial starter strains for increasing flavour and aroma attributes during winemaking.
This project has delivered a holistic view of the yeast and fungal microbiology that underpins wild fermentations undertaken across Australia. It has provided a methodology to provide winemakers with an ongoing means to investigate the unique biology of their fermentations, while outlining tools to alter wine characteristics and assist in fermentation efficiency. Selected non-Saccharomyces strains were also developed to provide options for the significant sectors of the Australian industry that wish to continue using commercial starter strains, but desire winemaking properties that go beyond those provided by standard wine yeast.