**4. Factors affecting the microbial communities' formation**

Grapevines' associated microbial communities originated from distinct geographic regions exhibit different profiles [13, 18, 34, 36, 55]. Each region is differentiated by the dominance of a few species per region. Indicatively, *Aspergillus* and *Penicillium* spp. were largely associated with the Chardonnay in Napa, while *Actinobacteria*,

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spoilage.

*Contribution of the Microbiome as a Tool for Estimating Wine's Fermentation Output…*

regulatory constraints, may explain the contradictory results.

most frequently isolated from Carignan (75%) [58].

agronomic practices [13, 35, 48, 52, 53].

*Bacteroides*, *Saccharomycetes*, *and Erysiphe necator* dominated in Central Coast, as well as *Proteobacteria* and *Botryotinia fuckeliana* in Sonoma [3]. Additionally, the prevalence of *Lachancea* in the Alentejo appellation was reported by Pinto et al. [13] while of *Rhodotorula* and *Botryotinia* was shown in the Estremadura appellation. Finally *Ramularia* and *Hanseniaspora* were the dominant genera in Bairrada, *Rhodotorula* and *Lachancea* in Dão, *Rhodotorula* and *Erysiphe* in Douro, and *Rhodotorula* and *Alternaria* in Minho appellation. Furthermore, the fungal grapes' associated diversity is also affected by agronomic practices. Vineyards that employed conventional, integrated pest management systems, organic, biodynamic, and ecophyto practices were shown to harbor different fungal communities [19, 23, 24, 44, 46, 48, 56–59]. However, the fact that these studies were carried out in vineyards from different countries (Austria, France, Italy, Spain, and Slovenia), subjected to different climates, pesticides, and

Many studies suggested that yeast diversity is dependent on climatic and microclimatic conditions. Higher yeast diversity has been described for vintages with high rainfall [40, 57] probably due to substantial fungal proliferation. Dry wines are produced by grapes submitted to prolonged withering in order to become moderately dried. The climate, as well as the extent of the withering period, was found to affect the formation of the fungal microbiome on grape skins in *V. vinifera* L. cv. Corvina, influencing the relative abundances of the fungal genera and consequently the secreted metabolites shaped in the must of Amarone red dry wine [57]. Grapes collected during a rainy season had increased bacterial biodiversity and enriched volatile compound (VOC) profile compared to a "dry" season collection, although some common microbial populations and VOC profiles maintained over the different vintages in grapes and musts samples, probably indicative of the typicity of Amarone.

Vineyard factors such as grape variety and berry chemical components are often described to influence microbial diversity [11, 43, 61, 62]. For instance, in similar soil and climatic conditions, *Cryptococcus* was the genera most frequently isolated (90% of all isolates) from Grenache grapes, whereas *Hanseniaspora* was the genus

The health status of berries can also affect the diversity of yeasts. The ascomycete *Botrytis cinerea* is considered one of the most damaging fungi in low temperature viticulture [60]. It causes *Botrytis* bunch rot, alternatively gray mold in grapes, affecting the physiochemical condition of grapes dramatically. Botrytized wine fermentations were found to contain increased abundance of acetic acid bacteria (AAB) in comparison with unaffected wines [61]. The elevated presence of AAB was additionally shown in botrytized wine fermentations obtained from the Dolce Winery, Oakville, California, analyzed via HTS [36]. Interestingly, the lactic acid bacteria (LAB) community was comprised mostly by *Leuconostoc* and *Lactococcus*, whereas *Oenococcus* was completely absent. Berries affected by *Botrytis cinerea* indicated increased development of the genus *Metschnikowia* [62]. Additionally, the bacterial community structure may vary depending on the grape cultivars or the

One of the factors found to contribute to microbial communities' formation is the amount of SO2. Comparison of the bacterial community dynamics following the fermentation process of hand-harvested organically grown Riesling grapes following organic and conventional *pied-de-cuve* (PDC) indicated that the species *Gluconobacter oxydans* was significantly affected by the addition of SO2 prior to PDC and bulk fermentation [37]. The ability of SO2 to prevent the growth of *Gluconobacter* at concentrations ≥25 mg/L was also shown by Bokulich and colleagues [63]. The elevated presence of this spoilage bacterium in organic fermentation highlights the susceptibility of the organic fermentation procedures to wine

*DOI: http://dx.doi.org/10.5772/intechopen.85692*

*Contribution of the Microbiome as a Tool for Estimating Wine's Fermentation Output… DOI: http://dx.doi.org/10.5772/intechopen.85692*

*Bacteroides*, *Saccharomycetes*, *and Erysiphe necator* dominated in Central Coast, as well as *Proteobacteria* and *Botryotinia fuckeliana* in Sonoma [3]. Additionally, the prevalence of *Lachancea* in the Alentejo appellation was reported by Pinto et al. [13] while of *Rhodotorula* and *Botryotinia* was shown in the Estremadura appellation. Finally *Ramularia* and *Hanseniaspora* were the dominant genera in Bairrada, *Rhodotorula* and *Lachancea* in Dão, *Rhodotorula* and *Erysiphe* in Douro, and *Rhodotorula* and *Alternaria* in Minho appellation. Furthermore, the fungal grapes' associated diversity is also affected by agronomic practices. Vineyards that employed conventional, integrated pest management systems, organic, biodynamic, and ecophyto practices were shown to harbor different fungal communities [19, 23, 24, 44, 46, 48, 56–59]. However, the fact that these studies were carried out in vineyards from different countries (Austria, France, Italy, Spain, and Slovenia), subjected to different climates, pesticides, and regulatory constraints, may explain the contradictory results.

Many studies suggested that yeast diversity is dependent on climatic and microclimatic conditions. Higher yeast diversity has been described for vintages with high rainfall [40, 57] probably due to substantial fungal proliferation. Dry wines are produced by grapes submitted to prolonged withering in order to become moderately dried. The climate, as well as the extent of the withering period, was found to affect the formation of the fungal microbiome on grape skins in *V. vinifera* L. cv. Corvina, influencing the relative abundances of the fungal genera and consequently the secreted metabolites shaped in the must of Amarone red dry wine [57]. Grapes collected during a rainy season had increased bacterial biodiversity and enriched volatile compound (VOC) profile compared to a "dry" season collection, although some common microbial populations and VOC profiles maintained over the different vintages in grapes and musts samples, probably indicative of the typicity of Amarone.

Vineyard factors such as grape variety and berry chemical components are often described to influence microbial diversity [11, 43, 61, 62]. For instance, in similar soil and climatic conditions, *Cryptococcus* was the genera most frequently isolated (90% of all isolates) from Grenache grapes, whereas *Hanseniaspora* was the genus most frequently isolated from Carignan (75%) [58].

The health status of berries can also affect the diversity of yeasts. The ascomycete *Botrytis cinerea* is considered one of the most damaging fungi in low temperature viticulture [60]. It causes *Botrytis* bunch rot, alternatively gray mold in grapes, affecting the physiochemical condition of grapes dramatically. Botrytized wine fermentations were found to contain increased abundance of acetic acid bacteria (AAB) in comparison with unaffected wines [61]. The elevated presence of AAB was additionally shown in botrytized wine fermentations obtained from the Dolce Winery, Oakville, California, analyzed via HTS [36]. Interestingly, the lactic acid bacteria (LAB) community was comprised mostly by *Leuconostoc* and *Lactococcus*, whereas *Oenococcus* was completely absent. Berries affected by *Botrytis cinerea* indicated increased development of the genus *Metschnikowia* [62]. Additionally, the bacterial community structure may vary depending on the grape cultivars or the agronomic practices [13, 35, 48, 52, 53].

One of the factors found to contribute to microbial communities' formation is the amount of SO2. Comparison of the bacterial community dynamics following the fermentation process of hand-harvested organically grown Riesling grapes following organic and conventional *pied-de-cuve* (PDC) indicated that the species *Gluconobacter oxydans* was significantly affected by the addition of SO2 prior to PDC and bulk fermentation [37]. The ability of SO2 to prevent the growth of *Gluconobacter* at concentrations ≥25 mg/L was also shown by Bokulich and colleagues [63]. The elevated presence of this spoilage bacterium in organic fermentation highlights the susceptibility of the organic fermentation procedures to wine spoilage.

*Advances in Grape and Wine Biotechnology*

*thermotolerans*, and *Streptomyces bacillaris* [44–47].

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* 

Overall, the fungal population at a phylum level is very similar and mainly com-

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*,

prised 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*,

**4. Factors affecting the microbial communities' formation**

Grapevines' associated microbial communities originated from distinct geographic regions exhibit different profiles [13, 18, 34, 36, 55]. Each region is differentiated by the dominance of a few species per region. Indicatively, *Aspergillus* and *Penicillium* spp. were largely associated with the Chardonnay in Napa, while *Actinobacteria*,

*Torulaspora*, and *Sporobolomyces* [3, 18, 19, 24, 34, 35, 48, 49].

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*Dyella*, and *Bacillus species* [35].

**Figure 1.** *Variables related to the microbiome formation.*

Generally, many of these variables (e.g., climatic conditions or cultivar) are interdependent and may be clustered into broad groups of effects **(Figure 1)**. The study of Bokulich and Mills [17] has shown that grape-associated microbial region is totally related with varietal, biogeographical, and climatic factors across multiscale viticultural zones. According to other study [20], the distribution of yeast species promotes significantly intra-vineyard spatial fluctuations. Continuously, the heterogeneity of grape samples harvested from single vineyards at the same stage of ripeness might be related, at least in part, to differing microbial communities in different sections of the vineyard. The biodiversity of yeast species in grapes is affected by numerous biotic and abiotic factors, as well as the interactions among the resident populations. However, more studies need to be performed in order to confidently elucidate the vineyard and grapevine phyllosphere microbiome.
