Applied Microbiology and Biotechnology

, Volume 102, Issue 2, pp 921–932 | Cite as

Application of directed evolution to develop ethanol tolerant Oenococcus oeni for more efficient malolactic fermentation

  • Alice L. Betteridge
  • Krista M. Sumby
  • Joanna F. Sundstrom
  • Paul R. Grbin
  • Vladimir JiranekEmail author
Applied microbial and cell physiology


Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This study aimed to use directed evolution (DE) to produce a more robust strain of O. oeni having the ability to withstand high ethanol concentrations. DE involves an organism mutating and potentially adapting to a high stress environment over the course of extended cultivation. A continuous culture of O. oeni was established and exposed to progressively increasing ethanol content such that after approximately 330 generations, an isolate from this culture was able to complete MLF in high ethanol content medium earlier than its parent. The ethanol tolerance of a single isolate, A90, was tested to confirm the phenotype and its fermentation performance in wine. In order to investigate the genotypic differences in the evolved strain that led to the ethanol tolerance phenotype, the relative expression of a number of known stress response genes was compared between SB3 and A90. Notably, there was increase in hsp18 expression in 20% (v/v) ethanol by both strains with A90 exhibiting a higher degree of expression. This study is the first to use directed evolution for O. oeni strain improvement and confirms that this technique can be used successfully for the development of new candidate strains for the wine industry. This study also adds to the current knowledge on the genetic basis of ethanol tolerance in this bacterium.


Oenococcus Wine Ethanol Stress response Directed evolution Malolactic fermentation 



VJ conceived the project. ALB performed the DE, screening of isolates and fermentation experiments, and analysed the data. KMS performed qPCR experiments, and analysed and interpreted the data. JFS performed strain passaging and phenotype stability experiments and analysed the data. ALB, KMS and JFS wrote and edited the manuscript. PRG and VJ revised and edited the manuscript.

Funding information

Strain SB3 was kindly donated by Laffort Australia PTY LTD. This research was supported by Australia’s grapegrowers and winemakers through their investment body, Wine Australia (formerly Australian Grape and Wine Authority), with matching funds from the Australian Government. The University of Adelaide is a member of the Wine Innovation Cluster. Part of VJ’s contribution to this work was made via ARC project IC130100005.

Compliance with ethical standards

Conflict of interest

ALB was supported by scholarships from AGWA (UA1101/P3102/GWR Ph 0901) and the University of Adelaide. KMS and JFS are supported by Wine Australia project funding (UA1302). The authors declare that they have no conflicts of interest.

Human and animal rights and informed consent

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2017_8593_MOESM1_ESM.pdf (620 kb)
ESM 1 (PDF 620 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Wine and Food Science, School of Agriculture, Food and WineUniversity of Adelaide, Waite CampusUrrbraeAustralia
  2. 2.Australian Research Council Training Centre for Innovative Wine ProductionUrrbraeAustralia

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