The relationship between grape juice composition and the progress of alcoholic and malolactic fermentation
Difficulties with alcoholic and malolactic fermentation are routinely reported, and can be attributed to a diverse range of causes. Poor fermentation progress can occur even in juices and wines that otherwise satisfy the usual criteria indicative of appropriate fermentation progress (e.g. YAN, Baumé, and SO2). Uncontrolled growth of non-target microorganisms has been reported to be inhibitory to alcoholic fermentation, either through consumption of nutrients, or through the production of secondary metabolites.
Sulfur dioxide additions to bins and crushers are used to control pre-fermentation microbial activity; however, even moderate levels of total SO2 can negatively affect the progress of malolactic fermentation. In addition, some yeasts produce large amounts of SO2 which is inhibitory to malolactic fermentation. This is a particular concern as simultaneous alcoholic and malolactic fermentations are increasingly being used to more efficiently manage scheduling issues associated with conducting malolactic fermentation as a separate process, after alcoholic-fermentation.
Clearly the areas of yeast and bacterial fermentation performance are inter-related, and understanding the risks and capturing opportunities of yeast/bacterial interactions requires an integrated approach as described in this project. Hence this project brings together two previously separate research areas, yeast and bacterial fermentation, in order to realise an integrated approach to the study of alcoholic and malolactic fermentation performance.
The proposed fermentation performance program will study the following:
- yeast/environment interactions, using the barcoded yeast collection to determine strain fitness and implantation efficiency, together with a survey of juice composition across multiple vintages, taking account of transport conditions and other harvest variables to determine their impact on composition (collaboration with Project 3.3.1)
- bacterial/environment interactions, by using model fermentations to identify factors that stimulate or inhibit malolactic fermentation, and through developing a transformation system for Oenococcus oeni to study genetic elements (inter-strain variable regions) and their effects on malic acid utilisation
- pilot and industry trials to evaluate the suitability of uniquely Australian regional isolates of malolactic bacteria, and to determine the robustness of co-inoculated fermentations using a range of winemaking interventions.
Quantifying the genetic response of Oenococcus oeni to SO2
It has been known for some time that high total SO2 concentrations are a primary factor in the failure of MLF. In response, considerable effort has been expended at the AWRI and elsewhere, exploring the physiological response of Oenococcus oeni to inoculation into fermentations or wine containing various concentrations of SO2. Strain-specific responses, or lack thereof, have been characterised and timing of co-inoculation has been optimised for O. oeni during alcoholic fermentation. While there is now an increasingly detailed understanding of the various factors that interact with SO2 to shape the likely success or failure of MLF, the genetic factors that might tip the balance in favour of the bacteria when it is challenged by SO2 concentrations are not understood.
To characterise the genetic response of O. oeni to SO2, and therefore gain insight into the survival strategies employed by this species, an experiment was undertaken where gene expression levels were quantified in response to an SO2 challenge. While still in its preliminary stages, the results of this work suggest that unlike many other wine-related microorganisms, O. oeni has few tools in its genetic toolbox that allow it to effectively deal with even low concentrations of SO2. This perhaps explains the extreme sensitivity of the organism to SO2 and reiterates that management of the environment into which O. oeni is inoculated is one of the key factors in ensuring a successful MLF.
Investigating interspecies microbial interactions
Considering the many different types of microorganisms present in grape juice at the start of fermentation and the competition for resources that likely occurs immediately following grape crushing, the ability of Saccharomyces cerevisiae to eventually dominate the environment is a testament to its robust biology. However, recent metagenomic profiling of uninoculated ferments at the AWRI and elsewhere shows that domination by S. cerevisiae is not an assured outcome. In addition, with non-Saccharomyces yeast being used increasingly as direct inocula into grape juice, often at high cell densities and usually prior to inoculation with S. cerevisiae, an obvious question to ask is whether some strains are better competitors than others? Are there strain-specific responses of S. cerevisiae to the presence of other organisms in their environment?
Following the establishment of a barcoded wine yeast collection at the AWRI, it was possible to experimentally approach the question of wine yeasts’ competitive response to grape juices that had previously been inoculated with different non-Saccharomyces yeast strains commonly found in grape juice or available as commercial preparations. With 94 strains in the collection, the question of whether some strains were better able to cope with the increased competition from non-Saccharomyces yeast could be addressed. Several specific S. cerevisiae/non-cerevisiae strain pairs were observed that did not appreciate the close contact with one another. These observations are currently being verified using a range of more specific experiments.