**3. Identification of the microbial communities**

*Terroir* is characterized by a multi-complex ecosystem where the vine (genetic material and cultural practices) interacts with the environmental factors (i.e., soil, climate, microclimate, humans, etc.) affecting the quality and typicity of the wine produced in a particular location. The understanding of the microbial terroir involves the identification of the microbes shaping grapes' environmental communities and the evaluation of their diversity dynamical evolution throughout the different stages of fermentation, until wine production. During natural fermentation the complex microbial communities that comprise the grape microbiome, including, yeasts, yeast-like fungi, and bacteria, are under the selective pressure of the

*Advances in Grape and Wine Biotechnology*

technological characteristics [9–11].

sensorial characteristics of PDO wines [14, 16].

**2. Methods to identify grape microbial species**

the process of fermentation has great influence in the nutritional, hygienic, safety, and organoleptic characteristics of the final product [2]. In a large number of fermented products, the formation of microbial biodiversity existing in the initial substrate is affected by a large number of factors, including the geographic origin, the cultural practices, differences among varieties, or the climatic conditions [3]. The contribution of the microbial community configuration, which is governed by spatial factors, land topography, environmental factors, etc. that sustain the spatial structure of the inhabitants, and their potential relation with the metabolic and sensorial characterizations of the final product, has been under deep research, leading to the suggestion of the microbial terroir [4]. The perspective of analyzing the microbial communities' dynamics as progressively differentiated during the process of fermentation for the determination of microbial terroir has been applied

in grapes and consequently its final fermented product, the wine [5, 6].

Traditionally winemaking process relies on spontaneous fermentation, which is conducted without the addition of chemical compounds or supplementary microbes at the beginning of the fermentation process. Under spontaneous fermentation conditions, the microbial community participating in fermentation and which is responsible for the quality of the final product is considered to be quite unpredictable. At the initial stages of fermentation, the microbial communities are comprised by a rich biodiversity of several yeast and mold species, including *Metschnikowia*, *Candida*, *Hanseniaspora*, *Pichia*, *Lachancea*, *Kluyveromyces*, and *Saccharomyces* [7–9]*.* During must fermentation, the alcoholic fermentation conducted elevates the ethanol content and establishes the basic fermenters, such as *Saccharomyces cerevisiae,* among the predominant species [9]. Their dominant presence during the fermentation process has led to the isolation of several *S. cerevisiae* strains, which have been extensively studied for the potential application of their

The development of high-throughput sequencing technologies has allowed the evaluation of the microbial consortium comprising grapes' microbiome in terms of revealing the concept of the microbial terroir [12–16]. The contribution of originassociated factors of grape varieties, including climate and microclimate, region site, as well as grape cultivar, in the microbial community formation and the final metabolic profiles, has been recently investigated [12, 17–20]. These studies have led to an improved spatial and temporal determination of the wine grapes' microbiome and brought new insights into its dynamics and biodiversity, revealing a new horizon for the better characterization of this product, especially in the case of PDO and PGI wines' designation. These labels were established by the European Union (EU) to guarantee the authentication of the local products produced in distinct geographic origin, applying traditional specialties. Metagenomic studies have been recently applied to identify the microbial communities that influence the original

The aim of this chapter is to extensively review all latest literature in the scope to investigate (a) how the microbiome communities are dynamically differentiated from grape to ready-to-drink wine, in order to finalize each wine's unique sensorial characteristics ,and (b) whether the microbiome could be used as a fingerprint tool for regional characterization, based on high-throughput sequencing (HTS) technologies.

Grapes are comprised by a complex microbiome, the members of which share different physiological characteristics and effects upon wine production. Some of them are present only in grapes and soil, such as parasitic fungi and environmental

**98**

alterations in the must microenvironment, caused by microbial interactions, as well as chemical and physical factors [39]. The must microbes have to handle stressful factors that affect their survival, including reduced oxygen, high ethanol and sulfur dioxide (SO2) levels, and low pH [40]. Moreover, the amounts of sugar existing in must favor for particular species, and high sugar content sweet wines select for osmotolerant species [41, 42]. As a consequence of this stressful microenvironment, numerous environmental species become unable to survive, while others, which are able to perform alcoholic fermentation and were detected in reduced relative abundance before fermentation, such as *Saccharomyces cerevisiae*, become dominant by the end of fermentation [16]. Apart from alcoholic fermentation, malolactic fermentation (MLF) (conversion of malic acid into lactic acid) is also involved in the metabolic transformation of grape juice into wine, conducted mostly by lactic acid bacteria (LAB), including the genera *Oenococcus*, *Lactobacillus*, *Pediococcus*, and *Leuconostoc*, leading to must deacidification, a process that affects organoleptic characteristics' formation [43]. By the end of fermentation, the microbial diversity is limited to selected microbial species [12, 35]. As revealed by several studies, some species were found to decline rapidly at the initial or the middle stages of fermentation, such as *Cryptococcus carnescens*, *Paraburkholderia terricola*, *Aureobasidium pullulans*, and *Metschnikowia pulcherrima*, while others exist until the end of fermentation, including *Saccharomyces cerevisiae, Torulaspora delbrueckii*, *Lachancea thermotolerans*, and *Streptomyces bacillaris* [44–47].

Overall, the fungal population at a phylum level is very similar and mainly comprised by *Ascomycota*, the most abundant phylum, followed by *Basidiomycota* [3, 18, 19, 24, 35, 48]. Additional phyla frequently detected but in limited concentrations include *Zygomycota* and *Chytridiomycota*. The most commonly found filamentous fungi genera include *Aspergillus*, *Erysiphe*, *Alternaria*, *Cladosporium*, *Penicillium*, *Davidiella*, *Lewia*, *Botrytis*, as well as the yeast-like fungus *Aureobasidium pullulans*. Further yeast genera commonly found include *Issatchenkia*, *Candida*, *Hanseniaspora*, *Pichia*, *Rhodotorula*, *Metschnikowia*, *Lachancea*, *Filobasidiella*, *Cryptococcus*, *Torulaspora*, and *Sporobolomyces* [3, 18, 19, 24, 34, 35, 48, 49].

High-throughput sequencing studies have been applied to evaluate the bacterial communities associated with the vineyard. The most frequently detected phyla in vineyard soils and grapevine roots include *Proteobacteria*, *Bacteroidetes*, *Acidobacteria*, *Verrucomicrobia*, *Planctomycetes*, *Actinobacteria*, *Chloroflexi*, *Gemmatimonatedes*, and *Firmicutes* [21, 50–52]. High-throughput analysis of the grapevine phyllosphere, flowers, and grape berry surface indicated that the bacterial communities were predominated by *Proteobacteria* followed by *Firmicutes*, *Actinobacteria*, *Acidobacteria*, and *Bacteroidetes* [13, 38, 53, 54]. The relative abundances of the groups may vary depending on the plant tissue or organ. The dominant taxa include members of the genera *Pseudomonas*, *Sphingomonas*, *Frigoribacterium*, *Curtobacterium*, *Bacillus*, *Enterobacter*, *Acinetobacter*, *Erwinia*, *Citrobacter*, *Pantoea*, and *Methylobacterium* [3, 13, 21, 48, 53, 54]. In contrast, the endophytic community in grape berries is mainly comprised by *Ralstonia*, *Burkholderia*, *Pseudomonas*, *Staphylococcus*, *Mesorhizobium*, *Propionibacterium*, *Dyella*, and *Bacillus species* [35].
