Taxonomic and Functional Characterization of the Microbial Community During Spontaneous in vitro Fermentation of Riesling Must Sirén, Kimmo; Mak, Sarah Siu Tze; Melkonian, Chrats; Carøe, Christian; Swiegers, Jan Hendrik; Molenaar, Douwe; Fischer, Ulrich; Gilbert, M. Thomas P. Published in: Frontiers in Microbiology DOI: 10.3389/fmicb.2019.00697 Publication date: 2019 Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Sirén, K., Mak, S. S. T., Melkonian, C., Carøe, C., Swiegers, J. H., Molenaar, D., Fischer, U., & Gilbert, M. T. P. (2019). Taxonomic and Functional Characterization of the Microbial Community During Spontaneous in vitro Fermentation of Riesling Must. Frontiers in Microbiology, 10, [697]. https://doi.org/10.3389/fmicb.2019.00697 Download date: 27. sep.. 2021 fmicb-10-00697 April 8, 2019 Time: 16:4 # 1 ORIGINAL RESEARCH published: 09 April 2019 doi: 10.3389/fmicb.2019.00697 Taxonomic and Functional Characterization of the Microbial Community During Spontaneous in vitro Fermentation of Riesling Must Kimmo Sirén1,2†, Sarah Siu Tze Mak3†, Chrats Melkonian4, Christian Carøe3, Jan Hendrik Swiegers5, Douwe Molenaar4, Ulrich Fischer1 and M. Thomas P. Gilbert3,6* 1 Institute for Viticulture and Oenology, Dienstleistungszentrum Ländlicher Raum Rheinpfalz, Neustadt an der Weinstraße, Germany, 2 Department of Chemistry, University of Kaiserslautern, Kaiserslautern, Germany, 3 Section for Evolutionary Genomics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark, 4 Systems Bioinformatics, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 5 Chr. Hansen A/S, Hoersholm, Denmark, 6 University Museum, Norwegian University of Science and Technology, Trondheim, Norway Although there is an extensive tradition of research into the microbes that underlie the Edited by: winemaking process, much remains to be learnt. We combined the high-throughput Rosanna Tofalo, University of Teramo, Italy sequencing (HTS) tools of metabarcoding and metagenomics, to characterize how Reviewed by: microbial communities of Riesling musts sampled at four different vineyards, and Irene Stefanini, their subsequent spontaneously fermented derivatives, vary. We specifically explored The University of Warwick, United Kingdom community variation relating to three points: (i) how microbial communities vary by Hervé Alexandre, vineyard; (ii) how community biodiversity changes during alcoholic fermentation; and (iii) Université de Bourgogne, France how microbial community varies between musts that successfully complete alcoholic *Correspondence: fermentation and those that become ‘stuck’ in the process. Our metabarcoding data M. Thomas P. Gilbert [email protected] showed a general influence of microbial composition at the vineyard level. Two of †These authors have contributed the vineyards (4 and 5) had strikingly a change in the differential abundance of equally to this work Metschnikowia. We therefore additionally performed shotgun metagenomic sequencing on a subset of the samples to provide preliminary insights into the potential relevance Specialty section: This article was submitted to of this observation, and used the data to both investigate functional potential and Food Microbiology, reconstruct draft genomes (bins). At these two vineyards, we also observed an increase a section of the journal Frontiers in Microbiology in non-Saccharomycetaceae fungal functions, and a decrease in bacterial functions Received: 17 January 2019 during the early fermentation stage. The binning results yielded 11 coherent bins, Accepted: 19 March 2019 with both vineyards sharing the yeast bins Hanseniaspora and Saccharomyces. Read Published: 09 April 2019 recruitment and functional analysis of this data revealed that during fermentation, a Citation: high abundance of Metschnikowia might serve as a biocontrol agent against bacteria, Sirén K, Mak SST, Melkonian C, Carøe C, Swiegers JH, Molenaar D, via a putative iron depletion pathway, and this in turn could help Saccharomyces Fischer U and Gilbert MTP (2019) dominate the fermentation. During alcoholic fermentation, we observed a general Taxonomic and Functional Characterization of the Microbial decrease in biodiversity in both the metabarcoding and metagenomic data. Unexpected Community During Spontaneous Micrococcus behavior was observed in vineyard 4 according to metagenomic analyses in vitro Fermentation of Riesling Must. based on reference-based read mapping. Analysis of open reading frames using Front. Microbiol. 10:697. doi: 10.3389/fmicb.2019.00697 these data showed an increase of functions assigned to class Actinobacteria in the Frontiers in Microbiology| www.frontiersin.org 1 April 2019| Volume 10| Article 697 fmicb-10-00697 April 8, 2019 Time: 16:4 # 2 Sirén et al. Shotgun and Metabarcoding Riesling Vineyards 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. Keywords: shotgun sequencing, metabarcoding, wine, microbial diversity, alcoholic fermentation INTRODUCTION remain of interest (Stefanini et al., 2016). It is currently understood that while microbial diversity decreases during Microbial interactions are vital to the winemaking process, the winemaking process (Bisson et al., 2017), some microbes with numerous different microbes known to be involved in can survive (Torija et al., 2001; Romano et al., 2003), such the formation of wine flavor and aroma. Understanding this as the yeast Metschnikowia pulcherrima (Díaz et al., 2018) microbial diversity and their interactions throughout the process and some bacteria such as Lactobacillus, Lactobacillaceae, and stands to enhance our knowledge of winemaking and wine Gluconobacter (Bokulich et al., 2015; Piao et al., 2015). Although complexity (Tempère et al., 2018). Although research on wine traditionally how the different microbial species interact has been microbes has a long history (Pasteur, 1872), many significant studied using culture-based techniques, they are increasingly challenges remain to be solved, not least due to difficulties targeted using culture-independent methods (Jolly et al., 2014; in studying the composition of wine’s complex matrix. In this Bagheri et al., 2017; Sternes et al., 2017; Morgan et al., 2018; regard, there is considerable interest in the application of high- Zepeda-Mendoza et al., 2018). throughput sequencing (HTS) tools such as metabarcoding and Another area of growing interest relates to the role of shotgun metagenomic sequencing to wine research, given their spontaneous fermentation. Because this is driven principally potential to offer us more in-depth characterization of the by non-Saccharomyces yeasts, spontaneous fermentations are microbial community (Belda et al., 2017; Stefanini and Cavalieri, regarded as being able to diversify aromatic quality (Zott 2018; Sirén et al., 2019). et al., 2011; Liu et al., 2016). However, their use remains Questions that have received considerable attention include intimidating for the industry, as they can lead to unwanted the origin of yeasts that drive the fermentation, and how different characteristics (Ciani et al., 2006), and sluggish, or even stuck, microbes shape the fermentation (Fleet, 1990, 2003; Pretorius, fermentations (Bisson, 1999; Bisson and Butzke, 2000). While the 2000; Bisson et al., 2017; Varela and Borneman, 2017). However, main reason for sluggish fermentation is often nutrient related, the answers to these questions are not clear cut. For example, additional microbial interactions could play a role, for example, it remains debated as to whether sufficient Saccharomyces by reducing nutrient availability. Recently, metabarcoding data cerevisiae is present in the vineyard (thus entering the must have been used to suggest that high species richness (including during pressing) to drive fermentation (Martini, 1993). This the presence of non-Saccharomyces yeasts) in must samples can question is particularly timely today, given the trend to return negatively affect the capability of Saccharomyces to carry out the to spontaneous fermentation during winemaking, for reasons fermentation (Boynton and Greig, 2016). If so, the addition of relating to both typicality as well as arguments that spontaneously sulfur (SO2) to the harvested grapes or must might be a means fermented wines gain in complexity due to the more diverse to allow desired winery microbes to dominate, by removing microbial interactions (Di Maro et al., 2007). Furthermore, the competition originating from unwanted vineyard microbes. relative importance of the vineyard versus winery flora during Although there is an increasing trend to apply HTS tools fermentation remains inconclusive, and little is known about to study wine microbiology, with few exceptions these have how the two interact