Abstract
Although there is an extensive tradition of research into the microbes that underlie the winemaking process, much remains to be learnt. We combined the high-throughput sequencing (HTS) tools of metabarcoding and metagenomics, to characterize how microbial communities of Riesling musts sampled at four different vineyards, and their subsequent spontaneously fermented derivatives, vary. We specifically explored community variation relating to three points: (i) how microbial communities vary by vineyard; (ii) how community biodiversity changes during alcoholic fermentation; and (iii) how microbial community varies between musts that successfully complete alcoholic fermentation and those that become ‘stuck’ in the process. Our metabarcoding data showed a general influence of microbial composition at the vineyard level. Two of the vineyards (4 and 5) had strikingly a change in the differential abundance of Metschnikowia. We therefore additionally performed shotgun metagenomic sequencing on a subset of the samples to provide preliminary insights into the potential relevance of this observation, and used the data to both investigate functional potential and reconstruct draft genomes (bins). At these two vineyards, we also observed an increase in non-Saccharomycetaceae fungal functions, and a decrease in bacterial functions during the early fermentation stage. The binning results yielded 11 coherent bins, with both vineyards sharing the yeast bins Hanseniaspora and Saccharomyces. Read recruitment and functional analysis of this data revealed that during fermentation, a high abundance of Metschnikowia might serve as a biocontrol agent against bacteria, via a putative iron depletion pathway, and this in turn could help Saccharomyces dominate the fermentation. During alcoholic fermentation, we observed a general decrease in biodiversity in both the metabarcoding and metagenomic data. Unexpected Micrococcus behavior was observed in vineyard 4 according to metagenomic analyses based on reference-based read mapping. Analysis of open reading frames using these data showed an increase of functions assigned to class Actinobacteria in the end of fermentation. Therefore, we hypothesize that bacteria might sit-and-wait until Saccharomyces activity slows down. Complementary approaches to annotation instead of relying a single database provide more coherent information true species. Lastly, our metabarcoding data enabled us to identify a relationship between stuck fermentations and Starmerella abundance. Given that robust chemical analysis indicated that although the stuck samples contained residual glucose, all fructose had been consumed, we hypothesize that this was because fructophilic Starmerella, rather than Saccharomyces, dominated these fermentations. Overall, our results showcase the different ways in which metagenomic analyses can improve our understanding of the wine alcoholic fermentation process.
Highlights
Microbial interactions are vital to the winemaking process, with numerous different microbes known to be involved in the formation of wine flavor and aroma
2.79 million reads were generated yielding 105 operational taxonomic units (OTUs), after all filtering this was reduced to 2.75 millions reads representing 72 OTUs that were retained for subsequent analyses (Supplementary Table S5)
One key difference is similar to that previously suggested in the first shotgun sequencing paper applied to wine samples, where a Metschnikowia abundance bias was found between shotgun analysis and ITS2 marker gene, with ITS2 marker gene overestimating (Sternes et al, 2017)
Summary
Microbial interactions are vital to the winemaking process, with numerous different microbes known to be involved in the formation of wine flavor and aroma. Research on wine microbes has a long history (Pasteur, 1872), many significant challenges remain to be solved, not least due to difficulties in studying the composition of wine’s complex matrix In this regard, there is considerable interest in the application of highthroughput sequencing (HTS) tools such as metabarcoding and shotgun metagenomic sequencing to wine research, given their potential to offer us more in-depth characterization of the microbial community (Belda et al, 2017; Stefanini and Cavalieri, 2018; Sirén et al, 2019). It remains debated as to whether sufficient Saccharomyces cerevisiae is present in the vineyard ( entering the must during pressing) to drive fermentation (Martini, 1993) This question is timely today, given the trend to return to spontaneous fermentation during winemaking, for reasons relating to both typicality as well as arguments that spontaneously fermented wines gain in complexity due to the more diverse microbial interactions (Di Maro et al, 2007). While some authors have suggested that the main contributors to fermentation originate from the vineyard flora (Bokulich et al, 2014, 2016; MorrisonWhittle and Goddard, 2018), others argue that the winery flora dominates (Stefanini et al, 2016; Ganucci et al, 2018)
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