Comparing the hierarchy of inter- and intra-species interactions with population dynamics of wine yeast cocultures

Abstract

In winemaking, the development of new fermentation strategies, such as the use of mixed starter cultures with Saccharomyces cerevisiae (Sc) yeast and non-Saccharomyces (NS) species, requires a better understanding of how yeasts interact, especially at the beginning of fermentation. Despite the growing knowledge on interactions between Sc and NS, few data are available on the interactions between different species of NS. It is furthermore still unclear whether interactions are primarily driven by generic differences between yeast species or whether individual strains are the evolutionarily relevant unit for biotic interactions. This study aimed at acquiring knowledge of the relevance of species and strain in the population dynamics of cocultures between five yeast species: Hanseniaspora uvarum, Lachancea thermotolerans, Starmerella bacillaris, Torulaspora delbrueckii and Sc. We performed cocultures between 15 strains in synthetic grape must and monitored growth in microplates. Both positive and negative interactions were identified. Based on an interaction index, our results showed that the population dynamics seemed mainly driven by the two species involved. Strain level was more relevant in modulating the strength of the interactions. This study provides fundamental insights into the microbial dynamics in early fermentation and contribute to the understanding of more complex consortia encompassing multiple yeasts trains.

Keywords: diversity; genetic modification; microbial interactions; non-Saccharomyces.

Figure 1.

Experimental layout of the mono- and coculture microplate cultures.

Figure 2.

Growth of the 15 monocultures (composed of 50% of fluorescently tagged cells and 50% of WT cells of the same strain) for the 5 species tested in this study: H. uvarum (Hu), L. thermotolerans (Lt), S. bacillaris (Sb), S. cerevisiae (Sc) and T. delbrueckii (Td). All growth curves are represented in grey, growth curves of all three strains of a species are represented in colored lines and species are separated in facets. Monocultures were done in biological triplicates. Curves were ploted using the Loess smoothing method from R tidyverse package.

Figure 3.

Growth parameters of the 15 monocultures: observed maximum population (maxOD), growth rate hours⁻¹ (r), area under the curve (AUC) and latency in hours (lat, time to reach OD600 = 0.25). r and AUC were computed using modelling from the GrowthCurver R package. Monocultures were done in biological triplicates. Letters a,b,c indicate the statistical group of the species. Species effect and strain effect were evaluated using a nested ANOVA: P-values of both fixed (species effect) and random effect (strain effect) are indicated for each growth parameter. H. uvarum (Hu), L. thermotolerans (Lt), S. bacillaris (Sb), S. cerevisiae (Sc) and T. delbrueckii (Td).

Figure 4.

Classification of the possible outcomes of co-cultures compared to monocultures. A: No interaction—co-culture relates to the average of both monocultures, B: Overyielding—coculture is greater than both monocultures, C: Underyielding—co-culture is worse than both monocultures, D: Little interaction—coculture significantly differs from the average of both monocultures but remains in the range of both. S1 = strain 1, S2 = S2, in dashed line = the calculated average of both monocultures.

Figure 5.

Examples of case B (left panel) and C (right panel). The only overyielding observed was for the growth rate of the coculture of H. uvarum 3221 and T. delbrueckii 3337. Underyielding was observed with the maximum population of the coculture of L. thermotolerans V7-21 and S. cerevisiae 59A. Four biological replicates were run for each cocultures.

Figure 6.

Heatmap of the AUC index: positive index means an overall higher growth of the coculture. * denotes cocultures whose AUC is significantly different from the average AUC of both monocultures. Colors of cluster and species, as well as addition of the * were edited manually from the PDF file. The original figure from R is available in the data repository.

Figure 7.

Population fold change after 24-hour growth in SGM425 for each strain (panel) in coculture with the strain indicated in the column. Colors of the dot correspond to the species of the strain in the column. * denotes significant difference of the fold change to 1 as tested by t-test.