DP_AEM student
Faculdade de Ciências da Universidade de Lisboa
Doctoral Program in Biology (FCUL)
Prof. Ana Mendes-Ferreira, Prof. Rogério Tenreiro and Prof. Nuno Pereira Mira

Phd Thesis

Fostering the use of non-Saccharomyces yeast strains as starter-cultures and biocontrol agents for wine industry

PhD grant reference: PD/BD/150587/2020 (February, 2020 - May, 2024)


The winemaking process may be viewed as a global process that starts at the vineyard, where the raw material is produced, goes through fermentation stages carried out by yeasts and lactic acid bacteria at the wineries, and in some cases requires ageing, before the process ends at the bottling step. This process occurs in non-sterile conditions, and is therefore susceptible to the action of the grapes and winery- resident microorganisms. Their uncontrolled development throughout the process may be responsible for negative impacts including:


- reductions in the yields of grapes and wines;

- organoleptic modifications that depreciate the quality of grapes and wines;

- irreversible modifications in the sanitary conditions of the raw material and wines, in the case of the filamentous fungi by the production of mycotoxins while in the case of yeasts and bacteria by the production of biogenic amines and ethyl carbamate [1].


In order to control their growth, winegrowers have at their disposal chemical fungicides that are usually efficient for the purpose. Nevertheless, during the last years, several negative aspects associated to their use have been pointed out. These include problems related to the consumers’ health and environmental issues, as well as problems related to the emergence of multi-resistant strains that may compromise their efficacy in the future, even when applying the maximum doses allowed by EU regulations [2]. Thus, alternatives to these traditional treatments have been actively sought [2, 3], and bio-based strategies, such as the use of antagonistic microorganisms, emerged as natural options to the partial or total replacement of the chemical agents.

Among the studied antagonists, several non-Saccharomyces yeasts (NSY) species have been suggested for application as they have exhibited antimicrobial activity against grapes-phytopathogens such as Botrytis cinerea [4, 5], and wine spoilage yeasts, including Dekkera bruxellensis [6, 7]. It is generally accepted that their antagonistic activity is the result of at least one of four key mechanisms, depending on the NSY specie/strain, target and process stage of application. These mechanisms can be classified as competition for nutrients and space; parasitism and secretion of hydrolases; antibiosis mediated by diffusible and volatile compounds and induction of the host defense mechanisms [8]. All these mechanisms have been demonstrated for several NSY species for applications in post-harvested grapes or at the vineyard against phytopathogenic fungi [8]. On the other hand, for application in musts and wines against spoilage yeasts and/or bacteria, antibiosis is the most studied and important mechanism, which generally involves the production of an extracellular killer toxin that displays antimicrobial activity under winemaking conditions against targeted species [9 , 10].

Non-Saccharomyces yeasts are naturally present in grapes and vines and their vast biodiversity in winemaking environments has been reported by several authors [11–13]. Despite these yeasts have always been seen as spoilage agents, in the recent years, the interest of winemaking industry has been directed to their use as they possess several interesting metabolic properties and enzymatic activities, that are absent in Saccharomyces cerevisiae and that may contribute to the improvement of wine flavour properties and wine quality in general [14]. Among these NSY, some have already been identified as potential biocontrol agents, such as Starmerella bacillaris [15] and Metschnikowia pulcherrima [6], which points out a potential double-role of these species in winemaking as wine aroma modulators and biocontrol agents.

In this line, the ultimate goal of this work is to uncover non-Saccharomyces yeast strains, featuring a double role as biocontrol agents and starter-cultures, and explore the metabolic and molecular mechanisms behind those interesting phenotypes for a rational application of these yeasts in wine industry.

In order to accomplish this goal, the work will be divided in three major tasks (Figure 1), each one associated to a specific objective. The first task consists in screening the antagonistic activity of an autochthonous non-Saccharomyces yeast collection against genera of non-biotrophic phytopathogenic fungi such as Aspergillus, Botrytis, Mucor and Penicillium.


Figure 1: PhD Programme schedule.  

Based on the results gathered in the Task 1, particular isolates will be selected for the next tasks considering:

- their ability to inhibit a wide range of phytopathogenic fungi species;

- the existence of variability of antagonistic activity within the tested genera;

- their ability to display antagonistic activity by the production of VOCs.


Next, in Task 2, it is intended to assess microbial interactions and uncover the mechanisms underlying the antagonistic activity of the selected NSY by using systems biology approaches (comparative genomics, metabolomics and transcriptomics).

The results gathered in Task 2 will be valuable to elaborate a methodology of application of these yeasts, which is going to be validated under real winemaking conditions in Task 3 in order to achieve the general objective of this PhD thesis.


This PhD project is aligned with UN_SDGs (2030 Agenda) Goal 2, as the exploitation of autochthonous yeast that are able to produce metabolites that are effective in controlling microbial spoilage, and are not toxic to environment and human health, as opposed to widely used chemical preservatives, represent a keystone in the quest of the goal of developing “more sustainable food production systems”. 


Funding: This PhD project will be supported by a scholarship of the FCT Doctoral Program in Applied and Environmental Microbiology (DP_AEM) and by ABCYeasts project (Norte 2020, ANI  39793) from the Microbiology and Biotechnology Group of BioISI (UTAD Pole). 



1.         du Toit, M., & Pretorius, I. S. (2000). Microbial Spoilage and Preservation of Wine: Using Weapons from Nature’s Own Arsenal -A Review. South African Journal of Enology & Viticulture, 21(1), 74–96. doi:10.21548/21-1-3559

2.             Pertot, I., Caffi, T., Rossi, V., Mugnai, L., Hoffmann, C., Grando, M. S., … Anfora, G. (2017). A critical review of plant protection tools for reducing pesticide use on grapevine and new perspectives for the implementation of IPM in viticulture. Crop Protection, 97, 70–84. doi:10.1016/j.cropro.2016.11.025

3.             Romanazzi, G., Lichter, A., Gabler, F. M., & Smilanick, J. L. (2012). Recent advances on the use of natural and safe alternatives to conventional methods to control postharvest gray mold of table grapes. Postharvest Biology and Technology, 63(1), 141–147. doi:10.1016/j.postharvbio.2011.06.013

4.             Cordero-Bueso, G., Mangieri, N., Maghradze, D., Foschino, R., Valdetara, F., Cantoral, J. M., & Vigentini, I. (2017). Wild grape-associated yeasts as promising biocontrol agents against Vitis vinifera fungal pathogens. Frontiers in Microbiology, 8(NOV). doi:10.3389/fmicb.2017.02025

5.             Calvo-Garrido, C., Usall, J., Torres, R., & Teixidó, N. (2017). Effective control of Botrytis bunch rot in commercial vineyards by large-scale application of Candida sake CPA-1. BioControl, 62(2), 161–173. doi:10.1007/s10526-017-9789-9

6.             Oro, L., Ciani, M., & Comitini, F. (2014). Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts. Journal of Applied Microbiology, 116(5), 1209–1217. doi:10.1111/jam.12446

7.             Comitini, F., Ingeniis De, J., Pepe, L., Mannazzu, I., & Ciani, M. (2004). Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts. FEMS Microbiology Letters, 238(1), 235–240. doi:10.1016/j.femsle.2004.07.040

8.             Spadaro, D., & Droby, S. (2016). Development of biocontrol products for postharvest diseases of fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends in Food Science and Technology, 47, 39–49. doi:10.1016/j.tifs.2015.11.003

9.             Ciani, M., Capece, A., Comitini, F., Canonico, L., Siesto, G., & Romano, P. (2016). Yeast interactions in inoculated wine fermentation. Frontiers in Microbiology, 7(APR), 1–7. doi:10.3389/fmicb.2016.00555

10.          Ciani, M., & Comitini, F. (2011). Non-Saccharomyces wine yeasts have a promising role in biotechnological approaches to winemaking. Annals of Microbiology, 61(1), 25–32. doi:10.1007/s13213-010-0069-5

11.          Binati, R. L., Innocente, G., Gatto, V., Celebrin, A., Polo, M., Felis, G. E., & Torriani, S. (2019). Exploring the diversity of a collection of native non- Saccharomyces yeasts to develop co-starter cultures for winemaking. Food Research International, 122(February), 432–442. doi:10.1016/j.foodres.2019.04.043

12.          Sirén, K., Mak, S. S. T., Melkonian, C., Carøe, C., Swiegers, J. H., Molenaar, D., … Thomas P Gilbert, M. (2019). Taxonomic and functional characterization of the microbial community during spontaneous in vitro fermentation of riesling must. Frontiers in Microbiology, 10(APR), 1–17. doi:10.3389/fmicb.2019.00697

13.          Sangorrín, M. P., Lopes, C. A., Jofré, V., Querol, A., & Caballero, A. C. (2008). Spoilage yeasts from Patagonian cellars: Characterization and potential biocontrol based on killer interactions. World Journal of Microbiology and Biotechnology, 24(7), 945–953. doi:10.1007/s11274-007-9557-6

14.          Jolly, N. P., Varela, C., & Pretorius, I. S. (2014). Not your ordinary yeast: Non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Research, 14(2), 215–237. doi:10.1111/1567-1364.12111

15.          Fernandes Lemos Junior, W. J., Bovo, B., Nadai, C., Crosato, G., Carlot, M., Favaron, F., … Corich, V. (2016). Corrigendum to: Biocontrol ability and action mechanism of starmerella bacillaris (synonym candida zemplinina) isolated from wine musts against gray mold disease agent botrytis cinerea on grape and their effects on alcoholic fermentation [Front. Microbiol. Frontiers in Microbiology, 7(SEP), 1–12. doi:10.3389/fmicb.2016.01499

16.          Nadai, C., Fernandes Lemos, W. J., Favaron, F., Giacomini, A., & Corich, V. (2018). Biocontrol activity of Starmerella bacillaris yeast against blue mold disease on apple fruit and its effect on cider fermentation. PLoS ONE, 13(9), 1–15. doi:10.1371/journal.pone.0204350


Host institution: Biosystems & Integrative Sciences Institute - Faculty of Sciences, University of Lisbon

Advisors: Professor Rogério Tenreiro; Professor Ana Mendes-Ferreira ; Professor Nuno P. Mira ; Professor Rui Malhó


Publication and Meetings

Esteves, M. (2021, March) - Fostering the use of non-Saccharomyces yeast strains as starter-cultures and biocontrol agents in wine industry". - Seminar presentation via zoom.

(2020, February) - participation in the 8th Workshop of the Doctoral Program in Applied and Environmental Microbiology at Campus de Gualtar, University of Minho, Braga.

Poster communication

Esteves, M., Lage, P., Mira, N. P., Tenreiro, R., Mendes-Ferreira, A. (2021, November) - " Characterization of the antagonistic potential of wine yeasts for biocontrol of phytopathogenic fungi in grapes. Poster presented online at Microbiotec21 Webconference-  Congress of Microbiology and Biotechnology 2021. 

(2020, February) - 1st Open Meeting COST action CA18113 EuroMicropH - understanding and exploiting the impacts of low pH on micro-organisms. Held in Instituto Superior Técnico, Lisbon. - member of the Organization Comittee.



“Advance R for Ecology and Evolutionary Biology”