**2. Study on the ecology of S***. cerevisiae* **in the Rioja qualified designation of origin**

#### **2.1 Material and methods**

#### *2.1.1 Sample collection*

Samples were taken from 11 wineries (A–K) located in three different sub-zones of the Rioja designation of origin (Rioja Alta, Rioja Oriental, and Rioja Alavesa)

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identified.

the following equation:

**2.2 Results and discussion**

*sub-zone*

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical…*

over a period of 3 or 4 consecutive years (**Figure 1**). None of the wineries studied had ever used a commercial starter yeast. Wineries A and B were new; wineries C,

Alcoholic fermentation (AF) was carried out by the destemming and crushing method in stainless steel (wineries A, B, C, D, and F) or wooden vats (winery H). In the other wineries, AF was carried out following the traditional carbonic maceration method (whole grape) in open concrete vats (wineries E, G, I, J, and K). The wines underwent spontaneous AF with the indigenous microbiota in all cases. In each winery, one fermentation tank was monitored in each year studied. The sampling was carried out 24 hours after vatting, in tumultuous AF (density

Samples collected in sterile bottles were taken to the laboratory and processed as follows: serial decimal dilutions were performed, and the samples were seeded onto plates containing a chloramphenicol glucose agar medium (CGA). The plates were incubated at 28°C for 48 h. Plates containing between 30 and 300 colonies were examined, and 10 colonies were randomly isolated from each CGA plate. The colonies were analyzed in order to identify *Saccharomyces* and non-*Saccharomyces* yeast and the clonal distribution of the *S. cerevisiae* by mitochondrial DNA (mtDNA) restriction analysis. Yeast cells were grown overnight in a culture of 5 ml YPD. DNA extraction and mtDNA restriction were determined by the method described by Querol and Barrio [12]. The DNA was digested with the restriction endonucleases Alu I, RSa I, and Hinf I, in accordance with the supplier's instructions (Boehringer Mannheim). The restriction fragments were separated by electrophoresis in agarose 1% gels and visualized on a UV transilluminator after ethidium bromide staining. The different clones isolated in each fermentation were named with the letter of

the cellar (A–K), followed by a Roman numeral and year of harvest (1–4).

I.D. <sup>=</sup> <sup>1</sup> <sup>−</sup> ∑3 *nj* (*nj* <sup>−</sup> 1) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

*cerevisiae* strains with the same electrophoretic profile.

tion took place after destemming and crushing the grapes.

The clonal variability of each fermentation was determined as a percentage of different *S. cerevisiae* genotypes compared to the total colonies *S. cerevisiae*

The index of diversity (I.D.) [13] was calculated with the different *S. cerevisiae* strains identified in the tumultuous and final stages of fermentation, according to

where N is the total number of *S. cerevisiae* strains and *nj* is the number of *S.* 

*2.2.1 Ecology of the yeast population during alcoholic fermentation in Rioja Oriental* 

In this sub-zone, four wineries were sampled (A–D). In all of them, the vinifica-

As expected, and concurring with previous studies on the ecology of alcoholic fermentation (FA) [14, 15], non-*Saccharomyces* yeasts were detected mainly in the first stages of the fermentation (data not shown). The rest of the yeasts isolated in this sub-zone (316 colonies) were identified as belonging to the *S. cerevisiae* species.

<sup>N</sup> (N <sup>−</sup> 1) (1)

E, and K were about 20 years old, while the others were over 50 years old.

*DOI: http://dx.doi.org/10.5772/intechopen.84870*

*2.1.2 Microbial analyses: strain typing of S. cerevisiae*

1025 g/L) and final AF.

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical… DOI: http://dx.doi.org/10.5772/intechopen.84870*

over a period of 3 or 4 consecutive years (**Figure 1**). None of the wineries studied had ever used a commercial starter yeast. Wineries A and B were new; wineries C, E, and K were about 20 years old, while the others were over 50 years old.

Alcoholic fermentation (AF) was carried out by the destemming and crushing method in stainless steel (wineries A, B, C, D, and F) or wooden vats (winery H). In the other wineries, AF was carried out following the traditional carbonic maceration method (whole grape) in open concrete vats (wineries E, G, I, J, and K). The wines underwent spontaneous AF with the indigenous microbiota in all cases.

In each winery, one fermentation tank was monitored in each year studied. The sampling was carried out 24 hours after vatting, in tumultuous AF (density 1025 g/L) and final AF.

#### *2.1.2 Microbial analyses: strain typing of S. cerevisiae*

Samples collected in sterile bottles were taken to the laboratory and processed as follows: serial decimal dilutions were performed, and the samples were seeded onto plates containing a chloramphenicol glucose agar medium (CGA). The plates were incubated at 28°C for 48 h. Plates containing between 30 and 300 colonies were examined, and 10 colonies were randomly isolated from each CGA plate. The colonies were analyzed in order to identify *Saccharomyces* and non-*Saccharomyces* yeast and the clonal distribution of the *S. cerevisiae* by mitochondrial DNA (mtDNA) restriction analysis. Yeast cells were grown overnight in a culture of 5 ml YPD. DNA extraction and mtDNA restriction were determined by the method described by Querol and Barrio [12]. The DNA was digested with the restriction endonucleases Alu I, RSa I, and Hinf I, in accordance with the supplier's instructions (Boehringer Mannheim). The restriction fragments were separated by electrophoresis in agarose 1% gels and visualized on a UV transilluminator after ethidium bromide staining.

The different clones isolated in each fermentation were named with the letter of the cellar (A–K), followed by a Roman numeral and year of harvest (1–4).

The clonal variability of each fermentation was determined as a percentage of different *S. cerevisiae* genotypes compared to the total colonies *S. cerevisiae* identified.

The index of diversity (I.D.) [13] was calculated with the different *S. cerevisiae* strains identified in the tumultuous and final stages of fermentation, according to the following equation:

$$\text{LD.} = \mathbf{1} - \frac{\sum \mathbf{3} \, n\_f \, (n\_f - 1)}{\mathbf{N} \, (\mathbf{N} - 1)} \tag{1}$$

where N is the total number of *S. cerevisiae* strains and *nj* is the number of *S. cerevisiae* strains with the same electrophoretic profile.

#### **2.2 Results and discussion**

### *2.2.1 Ecology of the yeast population during alcoholic fermentation in Rioja Oriental sub-zone*

In this sub-zone, four wineries were sampled (A–D). In all of them, the vinification took place after destemming and crushing the grapes.

As expected, and concurring with previous studies on the ecology of alcoholic fermentation (FA) [14, 15], non-*Saccharomyces* yeasts were detected mainly in the first stages of the fermentation (data not shown). The rest of the yeasts isolated in this sub-zone (316 colonies) were identified as belonging to the *S. cerevisiae* species.

*Advances in Grape and Wine Biotechnology*

yeasts in all the winegrowing regions of the world can cause a loss of typicity and have a negative effect on the biodiversity of natural yeasts present in the wineries [2]. *S. cerevisiae* is the predominant yeast species in alcoholic fermentation and the main element responsible for the characteristics of the wines. Many surveys carried out with spontaneous fermentations in different wine-producing regions have demonstrated that there is high genetic diversity within this species in each vinification [3]. However, in most cases, only a small number of strains of *S. cerevisiae* are dominant, mainly in the tumultuous and final fermentation stages, representing a

Earlier studies have shown that some strains of *S. cerevisiae* have been isolated in several consecutive years in the same winery, which is why some authors have suggested the term "winery effect" [4], and also some strains of these species were detected in different wineries of the same wine-producing area, suggesting that they were representative of a specific enological ecosystem [5, 6]. Knight et al. [7] even found specific genotypes from a particular region. These findings suggest that specific native *S. cerevisiae* strains could be associated with a *terroir* and have an influence on terroir-associated wine characteristics [8].These authors found a correlation between genotypic and phenotypic groups and the geographical origin of the strains, supporting the concept that there can be a microbial aspect to *terroir*. Nowadays, many wineries and regions are carrying out strain selection programs with yeasts isolated from their vineyard/winery ecosystems, based on the idea that these yeasts are better adapted to their musts, which have characteristics determined by the grape varieties and the *terroir* [9, 10]. Thus, the use of these typical strains as starter yeasts could provide wines with distinctive characteristics of a particular winery or region. For this reason, studying the existence of strains which are specific to one winery or enological area is very interesting for the wine industry [11]. Rioja is a wine region in Spain with qualified designation of origin status. It is subdivided into three sub-zones: Rioja Alta, Rioja Oriental, and Rioja Alavesa. Rioja Alta is located on the western edge of the region and at higher elevations with an Atlantic climate. Rioja Oriental, the eastern section, is strongly influenced by a Mediterranean climate which makes this area the warmest and driest part of the region. Rioja Alavesa, with a similar climate to Rioja Alta, produces different wines due to the relatively poor condition of the soil. Each sub-zone has its own character, which results in different wines derived from the different compositions and origins of the soils and the climate conditions. The study of the ecology and biodiversity of the yeast population during alcoholic fermentation is an interesting and important step in the research into, and understanding of, a winegrowing area and should be a step prior to the selection and subsequent employment of the yeasts isolated from that area as starters. The aim of this work was to study the ecology of spontaneous fermentations in 11 wineries from all over the Rioja Designation of Origin during 3–4 consecutive years in order to establish the existence of typical strains belonging to wineries, sub-zones, and regional ecosystems. The wineries under study were distributed throughout the three sub-zones of the Rioja designation of origin.

**2. Study on the ecology of S***. cerevisiae* **in the Rioja qualified designation** 

Samples were taken from 11 wineries (A–K) located in three different sub-zones of the Rioja designation of origin (Rioja Alta, Rioja Oriental, and Rioja Alavesa)

high percentage out of the total number of strains identified.

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**of origin**

**2.1 Material and methods**

*2.1.1 Sample collection*

**Figure 1.** *Location of the wineries in the three different sub-zones of the Rioja designation of origin.*

The clonal variability in this species was different depending on the winery and the year studied within each winery (**Table 1**). Thus, while in cellars A and B the total clonal variability was 13.8 and 21.8%, respectively, in the other two (C and D), it was around 35%. Because of the lower variability in the first two wineries, the index of diversity was also smaller.

A percentage of the isolated microbiota in each harvest had already been identified in previous years (**Table 1**). This percentage was higher in wineries A and B, while in the others, this proportion was lower. However, the evolution in the population of the strains during fermentation was similar in all the wineries, since in all of them the fermentation in general was carried out by one or two majority yeasts (**Table 2**) associated with a variable number of minority strains (**Table 3**). This majority strain(s) represented between 50 and 82% of the total isolated in each vinification (**Table 2**). The only exception was found in vintage 2 of cellar C, in which there were three major strains with a percentage of 14.3% each. Previous research on the *S. cerevisiae* population in spontaneous fermentations also showed the existence of a great diversity of strains. Among them, one or two represented more than 50% of the total [16, 17].

Nonetheless, only in wineries A and B, there was one common majority strain in the vinifications studied in the 3–4 years (it is highlighted in the same color in **Table 2**). The fact that the same strains were predominant in successive campaigns in the same winery has already been described in other works [4, 18], for which reason they considered that these yeasts were representative of the winery or the processing area. In contrast, in other studies, common yeasts were not found from one campaign to another or were only found in small proportions [19, 20].

The results obtained could suggest that in wineries A and B, there are yeasts characteristic of the winery that lead the fermentation, in proportions that vary depending on the characteristics of the vintage. However, in the other two wineries, the fermentations were carried out by a succession of strains, among which one or two were majority, but different from one vintage to another.

The main difference between the four wineries studied in the Rioja Oriental sub-zone was their age. While wineries A and B were new (this study began in its third and first harvest, respectively), cellar C had been producing wine for 15 years and D for 50. This means that the latter two had more complex ecosystems, which have been formed over successive harvests, as evidenced by the greater number of clones that participated in the fermentations and the higher diversity index (**Table 1**).

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**Table 1.**

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical…*

**A** 1 29 6 20.7 0.65 – –

**B** 1 – – – – – –

**C** 1 – – – – – –

**D** 1 – – – – – –

**Clonal variability (%)**

2 28 7 25.0 0.77 3 71.4 3 22 4 18.2 0.33 2 86.3 4 30 6 20.0 0.46 3 83.2

2 20 5 25.0 0.76 – – 3 30 9 30.0 0.84 2 36.6 4 28 8 28.6 0.77 3 63.8

2 21 11 53.4 0.94 **–** – 3 22 8 36.4 0.77 2 13.6 4 24 11 45.8 0.87 3 45.8

2 20 10 50.0 0.86 – – 3 22 5 22.7 0,58 1 9.1 4 20 8 40.0 0.82 2 10.0

**Index of diversity** **Genotypes detected in previous years Number %**

**Number of different clones**

**Total 109 15 13.8 0.64**

**Total 78 17 21.8 0.83**

**Total 67 25 37.3 0.92**

**Total 62 20 32.3 0.91**

*Clonal diversity of S. cerevisiae yeasts in Rioja Oriental wineries in 4 consecutive years (1–4).*

In a later study, carried out by our research group in cellar B [21], 72 colonies of the species *S. cerevisiae* were isolated in three fermentation tanks analyzed. The results showed 41 different clones, which provided a clonal variability of 56.9% and a biodiversity index of 0.98. These data would indicate that the fermentations were carried out by the succession of different clones and that there were no dominant ones. On the other hand, the comparison of the restriction profiles of this year's clones with those identified in the years specified in **Tables 1** and **2** showed that there was no correspondence with any of them, and therefore the majority strain (**Table 2**) that was isolated in the first three vintages as representative of the winery did not appear years later. Therefore, the ecology of the fermentations of this last year in cellar B was similar to those found in cellars C and D in the period shown previously. The situation in winery A could be similar, but it could not be proven since in the following years, commercial yeasts were inoculated, and the study could not be carried out. Taking into account the results shown, the presence of a dominant strain in the elaborations of wineries A and B could be due to the fact that

they are new wineries and not to the existence of representative yeasts.

On the other hand, the comparison of the different restriction profiles obtained in the four wineries during the 3–4 years of study showed that only two yeasts were common among the different wineries (**Table 3**). Therefore, the results obtained in this study did not show the existence of typical strain/s of this sub-zone. In the wine

*DOI: http://dx.doi.org/10.5772/intechopen.84870*

*S. cerevisiae* **analyzed**

**Winery Years Isolates of** 


*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical… DOI: http://dx.doi.org/10.5772/intechopen.84870*

#### **Table 1.**

*Advances in Grape and Wine Biotechnology*

index of diversity was also smaller.

**Figure 1.**

more than 50% of the total [16, 17].

The clonal variability in this species was different depending on the winery and the year studied within each winery (**Table 1**). Thus, while in cellars A and B the total clonal variability was 13.8 and 21.8%, respectively, in the other two (C and D), it was around 35%. Because of the lower variability in the first two wineries, the

A percentage of the isolated microbiota in each harvest had already been identi-

Nonetheless, only in wineries A and B, there was one common majority strain in the vinifications studied in the 3–4 years (it is highlighted in the same color in **Table 2**). The fact that the same strains were predominant in successive campaigns in the same winery has already been described in other works [4, 18], for which reason they considered that these yeasts were representative of the winery or the processing area. In contrast, in other studies, common yeasts were not found from one

The results obtained could suggest that in wineries A and B, there are yeasts characteristic of the winery that lead the fermentation, in proportions that vary depending on the characteristics of the vintage. However, in the other two wineries, the fermentations were carried out by a succession of strains, among which one or

The main difference between the four wineries studied in the Rioja Oriental sub-zone was their age. While wineries A and B were new (this study began in its third and first harvest, respectively), cellar C had been producing wine for 15 years and D for 50. This means that the latter two had more complex ecosystems, which have been formed over successive harvests, as evidenced by the greater number of clones that participated in the fermentations and the higher diversity

campaign to another or were only found in small proportions [19, 20].

two were majority, but different from one vintage to another.

fied in previous years (**Table 1**). This percentage was higher in wineries A and B, while in the others, this proportion was lower. However, the evolution in the population of the strains during fermentation was similar in all the wineries, since in all of them the fermentation in general was carried out by one or two majority yeasts (**Table 2**) associated with a variable number of minority strains (**Table 3**). This majority strain(s) represented between 50 and 82% of the total isolated in each vinification (**Table 2**). The only exception was found in vintage 2 of cellar C, in which there were three major strains with a percentage of 14.3% each. Previous research on the *S. cerevisiae* population in spontaneous fermentations also showed the existence of a great diversity of strains. Among them, one or two represented

*Location of the wineries in the three different sub-zones of the Rioja designation of origin.*

**120**

index (**Table 1**).

*Clonal diversity of S. cerevisiae yeasts in Rioja Oriental wineries in 4 consecutive years (1–4).*

In a later study, carried out by our research group in cellar B [21], 72 colonies of the species *S. cerevisiae* were isolated in three fermentation tanks analyzed. The results showed 41 different clones, which provided a clonal variability of 56.9% and a biodiversity index of 0.98. These data would indicate that the fermentations were carried out by the succession of different clones and that there were no dominant ones. On the other hand, the comparison of the restriction profiles of this year's clones with those identified in the years specified in **Tables 1** and **2** showed that there was no correspondence with any of them, and therefore the majority strain (**Table 2**) that was isolated in the first three vintages as representative of the winery did not appear years later. Therefore, the ecology of the fermentations of this last year in cellar B was similar to those found in cellars C and D in the period shown previously. The situation in winery A could be similar, but it could not be proven since in the following years, commercial yeasts were inoculated, and the study could not be carried out. Taking into account the results shown, the presence of a dominant strain in the elaborations of wineries A and B could be due to the fact that they are new wineries and not to the existence of representative yeasts.

On the other hand, the comparison of the different restriction profiles obtained in the four wineries during the 3–4 years of study showed that only two yeasts were common among the different wineries (**Table 3**). Therefore, the results obtained in this study did not show the existence of typical strain/s of this sub-zone. In the wine


*Clones with box in gray within the same winery indicate that they are the same strain, according to their mtDNA restriction profiles*

#### **Table 2.**

*Major S. cerevisiae strains in the fermentations of Rioja Oriental in 4 consecutive years (1–4).*


*Clones in bold and with box in gray in different columns show the same clones in different cellars*

#### **Table 3.**

*S. cerevisiae clones identified in each elaboration of Rioja Oriental in 4 consecutive years (1-4).*

area of Charentes in Cognac, a strain widely distributed throughout the area had been described, which was considered representative of the wine region. But unlike our data, this strain was the dominant one in all the samples where it appeared [18]. Likewise, in other ecological studies, widely disseminated strains were isolated in an area, and it was thought that they may be typical strains of that area [22].

**123**

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical…*

In this sub-zone, five wineries were sampled. Vinification was by both the destemming and crushing (F and H) and the carbonic maceration methods (E, G, and I). As happened in the Rioja Oriental, alcoholic fermentation in the Rioja Alta sub-zone was carried out by yeasts of the *S. cerevisiae* species, since the non-*Saccharomyces* group was identified mainly in the early stages of the process. However, in the third harvest of cellars E and F, three colonies belonging to non-*Saccharomyces* genera were isolated in tumultuous fermentation (data not shown). The detection of these yeasts in advanced stages of winemaking had also been reported in other works

*2.2.2 Ecology of the yeast population during alcoholic fermentation in Rioja Alta* 

[23, 24]. In total, 450 yeasts belonging to the *S. cerevisiae* species were studied.

Clonal variability was high in this sub-zone in all the wineries (**Table 4**). From the 450 isolated colonies of *S. cerevisiae*, 177 different clones were identified, resulting in high clonal variability in Rioja Alta, which ranged between 35% in winery H and 39% in winery F, with the exception of cellar I, in which 50% was reached. It is noteworthy that clonal variability depended on the year analyzed, being generally lower the first year and higher the third. These results could be related to the

The index of diversity was high and similar for all the wineries with a value of 0.95–0.96. These results were due to the high number of different strains that participated in each fermentation, which in the case of wineries F and I was favored by the low presence of common clones in different campaigns within the same winery (**Table 4**). In wineries E, G, and H, although there were more strains that appeared in different campaigns, they did so in a low percentage, and, therefore, the index remained high. The population evolution in this sub-zone was similar to Rioja Oriental, since the alcoholic fermentation was carried out by different clones during the fermentation process. In most of the vinifications, 1–3 majority clones were detected, representing at least 43% of the population (**Table 5**). However, in this sub-zone certain exceptions were found. Thus, in the first year analyzed in winery F, one clone represented 65% of the total and so was therefore dominant and responsible for the fermentation. This could be due to the special characteristics of this campaign (frosts at the end of April, high rainfall and strong winds in spring, hail storms and low temperatures in summer) that negatively influenced the grape ripening and meant that few yeasts were able to adapt and develop to carry out the fermentation. These conditions were more adverse in winery F, due to its geographic situation. It is

the winery located at higher altitude and closer to the mountains.

On the other hand, in wineries G and I, there were vinification processes in which no major clones were isolated, which could be related to the high number of different clones that participated in the fermentations. What these wineries have in common is that they follow the carbonic maceration method and have been making wine for more than 100 years. Santamaría et al. [15] showed that the number of different S*. cerevisiae* strains and the frequency of their appearance varied according to age. When comparing the clones found in the five wineries (**Table 6**), only pattern I, coming from the last year studied in the four wineries, was common in all of them. When comparing the restriction profiles of the four strains, all of them showed the

The presence of the same strain in four wineries could be due to the fact that the villages where wineries G, H, and I are located are geographically very close to each other (**Figure 1**). In cellar F, a little further away, a part of the grapes came from the same area as the other three. In this fourth year of study, strong winds were reported by grape growers in September and October. These winds could be responsible for transporting yeasts from one area to another, which would explain the appearance

*DOI: http://dx.doi.org/10.5772/intechopen.84870*

climatological characteristics of the harvest.

same electrophoretic pattern (**Figure 2**).

*sub-zone*

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical… DOI: http://dx.doi.org/10.5772/intechopen.84870*

## *2.2.2 Ecology of the yeast population during alcoholic fermentation in Rioja Alta sub-zone*

In this sub-zone, five wineries were sampled. Vinification was by both the destemming and crushing (F and H) and the carbonic maceration methods (E, G, and I).

As happened in the Rioja Oriental, alcoholic fermentation in the Rioja Alta sub-zone was carried out by yeasts of the *S. cerevisiae* species, since the non-*Saccharomyces* group was identified mainly in the early stages of the process. However, in the third harvest of cellars E and F, three colonies belonging to non-*Saccharomyces* genera were isolated in tumultuous fermentation (data not shown). The detection of these yeasts in advanced stages of winemaking had also been reported in other works [23, 24]. In total, 450 yeasts belonging to the *S. cerevisiae* species were studied.

Clonal variability was high in this sub-zone in all the wineries (**Table 4**). From the 450 isolated colonies of *S. cerevisiae*, 177 different clones were identified, resulting in high clonal variability in Rioja Alta, which ranged between 35% in winery H and 39% in winery F, with the exception of cellar I, in which 50% was reached. It is noteworthy that clonal variability depended on the year analyzed, being generally lower the first year and higher the third. These results could be related to the climatological characteristics of the harvest.

The index of diversity was high and similar for all the wineries with a value of 0.95–0.96. These results were due to the high number of different strains that participated in each fermentation, which in the case of wineries F and I was favored by the low presence of common clones in different campaigns within the same winery (**Table 4**). In wineries E, G, and H, although there were more strains that appeared in different campaigns, they did so in a low percentage, and, therefore, the index remained high.

The population evolution in this sub-zone was similar to Rioja Oriental, since the alcoholic fermentation was carried out by different clones during the fermentation process. In most of the vinifications, 1–3 majority clones were detected, representing at least 43% of the population (**Table 5**). However, in this sub-zone certain exceptions were found. Thus, in the first year analyzed in winery F, one clone represented 65% of the total and so was therefore dominant and responsible for the fermentation. This could be due to the special characteristics of this campaign (frosts at the end of April, high rainfall and strong winds in spring, hail storms and low temperatures in summer) that negatively influenced the grape ripening and meant that few yeasts were able to adapt and develop to carry out the fermentation. These conditions were more adverse in winery F, due to its geographic situation. It is the winery located at higher altitude and closer to the mountains.

On the other hand, in wineries G and I, there were vinification processes in which no major clones were isolated, which could be related to the high number of different clones that participated in the fermentations. What these wineries have in common is that they follow the carbonic maceration method and have been making wine for more than 100 years. Santamaría et al. [15] showed that the number of different S*. cerevisiae* strains and the frequency of their appearance varied according to age.

When comparing the clones found in the five wineries (**Table 6**), only pattern I, coming from the last year studied in the four wineries, was common in all of them. When comparing the restriction profiles of the four strains, all of them showed the same electrophoretic pattern (**Figure 2**).

The presence of the same strain in four wineries could be due to the fact that the villages where wineries G, H, and I are located are geographically very close to each other (**Figure 1**). In cellar F, a little further away, a part of the grapes came from the same area as the other three. In this fourth year of study, strong winds were reported by grape growers in September and October. These winds could be responsible for transporting yeasts from one area to another, which would explain the appearance

*Advances in Grape and Wine Biotechnology*

**122**

**Table 3.**

*restriction profiles*

**Table 2.**

area of Charentes in Cognac, a strain widely distributed throughout the area had been described, which was considered representative of the wine region. But unlike our data, this strain was the dominant one in all the samples where it appeared [18]. Likewise, in other ecological studies, widely disseminated strains were isolated in an area, and it was thought that they may be typical strains of that area [22].

*Clones in bold and with box in gray in different columns show the same clones in different cellars*

*S. cerevisiae clones identified in each elaboration of Rioja Oriental in 4 consecutive years (1-4).*

**Winery A Winery B Winery C Winery D**

*Major S. cerevisiae strains in the fermentations of Rioja Oriental in 4 consecutive years (1–4).*

**Years 1 2 3 4**

A-II1 24.1 A-II2 21.4

**Winery Strain % Strain % Strain % Strain % A** A-I1 55.2 A-I2 42.9 A-IV3 81.8 A-I4 73.3

**B** – – B-I2 35.0 B-I3 33.3 B-II4 21.4

**C** – – C-IV2 14.3 C-I3 40.9 C-I4 33.3

**D** – – D-V2 35.0 D-II3 63.6 D-I4 35.0

*Clones with box in gray within the same winery indicate that they are the same strain, according to their mtDNA* 

C-X2 14.3

**Total 79.3 64.3 81.8 73.3**

– – B-IV2 35.0 B-VIII3 20.0 B-IV4 42.3 **Total** – **70.0** 53.3 **63.7**

– – C-V2 14.3 C-IV3 27.3 C-IX4 16.7

**Total** – **42.9 68.2 50.0**

– – D-VI2 15.0 D-III4 25.0 **Total** – **50.0 63.6 60.0**

**1 2 3 4 2 3 4 2 3 4 2 3 4** A-I1 A-I2 A-I3 A-I4 **B-I2** B-I3 B-I4 C-I2 C-I3 C-I4 D-I2 D-I3 D-I4 A-II1 **A-II2** A-II3 **A-II4** B-II2 B-II3 B-II4 C-II2 C-II3 **C-II4** D-II2 D-II3 D-II4 A-III1 A-III2 A-III3 A-III4 B-III2 B-III3 B-III4 C-III2 C-III3 C-III4 D-III2 D-III3 D-III4 A-IV1 A-IV2 A-IV3 A-IV4 B-IV2 B-IV3 B-IV4 C-IV2 C-IV3 C-IV4 D-IV2 D-IV3 D-IV4 A-V1 A-V2 *A-V4* B-V2 B-V3 B-V4 C-V2 C-V3 C-V4 D-V2 D-V3 D-V4 A-VI1 A-VI2 A-VI4 B-VI3 **B-VI4** C-VI2 C-VI3 C-VI4 D-VI2 D-VI4 A-VII2 B-VII3 B-VII4 C-VII2 C-VII3 C-VII4 D-VII2 D-VII4

B-VIII3 B-VIII4 C-VIII2 C-VIII3 C-VIII4 D-VIII2 D-VIII4

C-X2 C-X4 D-X2

C-XI2 C-XI4

B-IX3 C-IX2 C-IX4 D-IX2


#### *Advances in Grape and Wine Biotechnology*

#### **Table 4.**

*Clonal diversity of S. cerevisiae yeasts in Rioja Alta wineries in 4 consecutive years (1–4).*

of the same strain in the four wineries. This yeast could come from the grapes, since in none of the three previous vintages had this strain been isolated in any of the cellars. This strain dominated the beginning of the fermentations and was later replaced by other indigenous strains, since in no case was it isolated at the end of the fermentation processes. These results would concur with those of Schütz and Gafner [19], who consider that the population of yeasts can be considered dependent on the harvest and the vineyard, and also with Le Jeune et al. [25], who indicated that the populations involved in spontaneous alcoholic fermentation result from a balance between the *S. cerevisiae* strains present in the grape and in the cellar.

The data obtained would show the existence of extensive microbiota in each winery, which, together with the microflora that accompanies the grapes, will develop according to the characteristics of the harvest. The fermentations would be the result of the sequence of different yeasts, and despite having found a widely spread yeast in the sub-zone 1 year, it did not appear in any other winery in the

**125**

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical…*

**Years 1 2 3 4**

**Winery Strain % Strain % Strain % Strain % E** E-I1 40.0 E-I2 13.3 E-I3 25.0 E-I4 20.0

**F** F-III1 65.0 F-II2 13.0 F-I3 25.0 F-I4 45.0

**G** G-I1 20.0 G-V2 20.0 G-I3 14.3 G-I4 33.3

**H** H-II1 25 H-III2 20.0 H-I3 30.0 H-I4 43.3

**I** – – I-I2 18.2 I-XV3 15.0 I-I4 50.0 I-IV2 36.4

F-VIII2 17.4

E-II1 20.0 E-IV2 16.7 E-III3 25.0 E-IV4 25.0

**Total 60.0 43.3 50.0 65.0**

F-III2 21.8 F-V3 30.0

**Total 65.0 52.2 55.0 45.0**

**Total 20.0 50.0 42.9 70.7**

H-III1 20 H-X2 13.3 H-II3 20.0 H-XI4 13.3 **Total 55 33.3 50.0 56.6**

**Total** – **54.6 15.0 50.0**

E-XI2 13.3 E-V4 20.0

G-VII2 15.0 G-IV3 28.6 G-III4 20.0 G-VII2 15.0 G-VI4 17.4

other three campaigns studied. So, we do not consider that in Rioja Alta, there are

In this sub-zone, two wineries (J and K) were sampled, where wines were produced by the carbonic maceration method. As in the other two sub-zones, the non-*Saccharomyces* yeasts participated at the beginning of the alcoholic fermentation, and later they were replaced by *S. cerevisiae* strains, which were the ones that directed and carried out the process until the end. In total, 149 colonies of these

The level of clonal variability was very high in both cellars, especially in winery K, where it reached 60% (**Table 7**). It is noteworthy that the highest percentage of clonal variability in both wineries was obtained in the third year, as in the vinifications in the Rioja Alta, which confirms the importance of the characteristics of the

As in the rest of the sub-zones, the high number of different clones identified in each fermentation and the few common yeasts between campaigns (**Table 7**) provided high indexes of diversity, which reached values of 0.95 and 0.98 for cellars J and K, respectively. These indices were of the same order as those found in Rioja Alta and higher than those of Rioja Oriental. Likewise, the number of yeasts that participated in more than one campaign was small in the two wineries, with the

*2.2.3 Ecology of the yeast population during alcoholic fermentation in the Rioja* 

representative strains from either the wineries or the sub-zone.

*Major S. cerevisiae strains in the fermentations of Rioja Alta in 4 consecutive years (1–4).*

harvest in the microbiota that drives the fermentations.

*Alavesa sub-zone*

**Table 5.**

species were isolated.

*DOI: http://dx.doi.org/10.5772/intechopen.84870*


*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical… DOI: http://dx.doi.org/10.5772/intechopen.84870*

#### **Table 5.**

*Advances in Grape and Wine Biotechnology*

*S. cerevisiae* **analyzed**

**Number of different clones**

**Total 92 34 37.0 0.96 F** 1 20 5 25.0 0.57 –

**Total 82 32 39.0 0.95**

**Total 104 40 38.5 0.95**

**Total 100 35 35.0 0.95**

**Total 72 36 50.0 0.96**

*Clonal diversity of S. cerevisiae yeasts in Rioja Alta wineries in 4 consecutive years (1–4).*

**G** 1 30 13 43.3 0.93 – –

**H** 1 20 9 45.0 0.88 – –

**I** 1 – – – – – –

**E** 1 30 10 33.3 0.80 – –

**Clonal variability (%)**

2 30 14 46.7 0.93 2 6.6 3 12 8 66.7 0.91 3 41.6 4 20 8 40.0 0.87 1 5.0

2 23 10 43.5 0.91 0 0 3 19 9 47.4 0.85 0 0 4 20 9 45.0 0.79 1 5.0

2 23 11 47.8 0.92 0 0 3 21 12 57.1 0.92 5 57.3 4 30 12 40.0 0.85 3 40.0

2 30 13 43.3 0.92 3 36.6 3 20 11 55.0 0.88 4 50.0 4 30 13 43.3 0.80 4 23.2

2 22 9 40.9 0.82 – – 3 30 20 66.7 0.97 3 13.3 4 20 11 55.0 0.76 1 5.0

**Index of diversity** **Genotypes detected in previous years Number %**

**Winery Years Isolates of** 

of the same strain in the four wineries. This yeast could come from the grapes, since in none of the three previous vintages had this strain been isolated in any of the cellars. This strain dominated the beginning of the fermentations and was later replaced by other indigenous strains, since in no case was it isolated at the end of the fermentation processes. These results would concur with those of Schütz and Gafner [19], who consider that the population of yeasts can be considered dependent on the harvest and the vineyard, and also with Le Jeune et al. [25], who indicated that the populations involved in spontaneous alcoholic fermentation result from a balance

The data obtained would show the existence of extensive microbiota in each winery, which, together with the microflora that accompanies the grapes, will develop according to the characteristics of the harvest. The fermentations would be the result of the sequence of different yeasts, and despite having found a widely spread yeast in the sub-zone 1 year, it did not appear in any other winery in the

between the *S. cerevisiae* strains present in the grape and in the cellar.

**124**

**Table 4.**

*Major S. cerevisiae strains in the fermentations of Rioja Alta in 4 consecutive years (1–4).*

other three campaigns studied. So, we do not consider that in Rioja Alta, there are representative strains from either the wineries or the sub-zone.

## *2.2.3 Ecology of the yeast population during alcoholic fermentation in the Rioja Alavesa sub-zone*

In this sub-zone, two wineries (J and K) were sampled, where wines were produced by the carbonic maceration method. As in the other two sub-zones, the non-*Saccharomyces* yeasts participated at the beginning of the alcoholic fermentation, and later they were replaced by *S. cerevisiae* strains, which were the ones that directed and carried out the process until the end. In total, 149 colonies of these species were isolated.

The level of clonal variability was very high in both cellars, especially in winery K, where it reached 60% (**Table 7**). It is noteworthy that the highest percentage of clonal variability in both wineries was obtained in the third year, as in the vinifications in the Rioja Alta, which confirms the importance of the characteristics of the harvest in the microbiota that drives the fermentations.

As in the rest of the sub-zones, the high number of different clones identified in each fermentation and the few common yeasts between campaigns (**Table 7**) provided high indexes of diversity, which reached values of 0.95 and 0.98 for cellars J and K, respectively. These indices were of the same order as those found in Rioja Alta and higher than those of Rioja Oriental. Likewise, the number of yeasts that participated in more than one campaign was small in the two wineries, with the


*Advances in Grape and Wine Biotechnology*

**Table 6.**

**127**

**Figure 2.**

**J**

**K**

**Table 7.**

*Alta wineries in year 4.*

**Winery Years Isolates of** 

*S. cerevisiae* **analyzed**

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical…*

*Restriction patterns obtained with different restriction endonucleases, from the patterns I isolated in four Rioja* 

**Clonal variability (%)**

**Index of diversity** **Genotypes detected in previous years**

3 29.9

5 50.0

3 15.0

**%**

–

–

0

**Number**

–

–

0

**Number of different clones**

1 29 15 51.7 0.93

2 30 13 43.3 0.92

3 20 14 70.0 0.94

1 20 12 60.0 0.95

2 30 14 46.7 0.92

3 20 19 95.0 0.99

**Total 70 42 60.0 0.98**

*Clonal diversity of S. cerevisiae yeasts in Rioja Alavesa wineries in 3 consecutive years (1–3).*

**Total 79 34 43.0 0.95**

exception of the third year of study in winery J. Therefore, the microbiota respon

responding to the fermentations of the second year. Therefore, the data obtained would show the existence of extensive microbiota in each winery. The fermenta

tions would be the result of the sequence of different populations of yeasts, as there

Regarding the population dynamics of the fermentation, it was observed that in the two wineries, the fermentations were carried out by different *S. cerevisiae* strains that followed each other during the different fermentative phases. In each vinification process, several major clones were detected, which represented at least 30% of the yeasts that carried out the fermentation (**Table 8**). The exception was the third year, in which most of the strains that were found in a fermentative phase were replaced by others in the following stage, particularly in winery K. This fact was also found in some of the vinifications in wineries G and I in Rioja Alta. All four wineries are over a hundred years old and conduct vinifications using the carbonic maceration method. The comparison of the different strains found in the two cellars during the 3 years of study (**Table 9**) showed the existence of a single common clone, cor

sible for the fermentation was different in each harvest.

are no representative strains of the sub-zone.




*DOI: http://dx.doi.org/10.5772/intechopen.84870*

*Biodiversity of Saccharomyces cerevisiae Yeasts in Spontaneous Alcoholic Fermentations: Typical… DOI: http://dx.doi.org/10.5772/intechopen.84870*

#### **Figure 2.**

*Advances in Grape and Wine Biotechnology*

**126**

**Winery E**

**1** E-I1 E-II1 E-III1 E-IV1

E-V1 E-VI1 E-VII1 E-VIII1

E-IX1

E-X1

E-X2 E-XI2 E-XII2 E-XIII2 E-XIV2

E-IX2

E-VIII2

E-VIII3

E-VIII4

F-VIII2

F-IX2 F-X2

F-IX3

F-IX4

G-IX1

G-X1 G-XI1 G-XII1 G-XIII1

G-XII3

G-XII4

H-XII2 H-XIII2

H-XIII4

H-XII4

G-XI2

G-XI3

G-XI4

H-XI2

H-XI3

H-XI4

G-X2

G-X3

G-X4

H-X2

H-X3

H-X4

G-IX2

G-IX3

G-IX4

H-IX1

H-IX2

H-IX3

H-IX4

I-IX2

I-IX3

I-X3 I-XI3 I-XII3

I-XIII3

I-XIV3

I-XV3

I-XVI3

I-XVII3

I-XVIII3

I-XIX3

I-XX3

*Clones with box in gray in different columns show the same clone in different cellars*

*S. cerevisiae clones identified in each elaboration of Rioja Alta in 4 consecutive years (1–4).*

**Table 6.**

II-XI4

II-X4

II-IX4

F-VIII3

F-VIII4

G-VIII1

G-VIII2

G-VIII3

G-VIII4

H-VIII1

H-VIII2

H-VIII3

H-VIII4

I-VIII2

I-VIII3

II-VIII4

E-VII2

E-VII3

E-VII4

F-VII98

F-VII3

F-VII4

G-VII1

G-VII2

G-VII3

G-VII4

H-VII1

H-VII2

H-VII3

H-VII4

I-VII2

I-VII3

II-VII4

E-VI2

E-VI3

E-VI4

F-VI2

F-VI3

F-VI4

G-VI1

G-VI2

G-VI3

G-VI4

H-VI1

H-VI2

H-VI3

H-VI4

I-VI2

I-VI3

II-VI4

E-V2

E-V3

E-V4

F-V1

F-V2

F-V3

F-V4

G-V1

G-V2

G-V3

G-V4

H-V1

H-V2

H-V3

H-V4

I-V2

I-V3

II-V4

E-IV2

E-IV3

E-IV4

F-IV1

F-IV2

F-IV3

F-IV4

G-IV1

G-IV2

G-IV3

G-IV4

H-IV1

H-IV2

H-IV3

H-IV4

I-IV2

I-IV3

II-IV4

E-III2

E-III3

E-III4

F-III1

F-III2

F-III3

F-III4

G-III1

G-III2

G-III3

G-III4

H-III1

H-III2

H-III3

H-III4

I-III2

I-III3

II-III4

E-II2

E-II3

E-II4

F-II1

F-II2

F-II3

F-II4

G-II1

G-II2

G-II3

G-II4

H-II1

H-II2

H-II3

H-II4

I-II2

I-II3

II-II4

E-I2

E-I3

E-I4

F-I1

F-I2

F-I3

F-I4

G-I1

G-I2

G-I3

G-I4

H-I1

H-I2

H-I3

H-I4

I-I2

I-I3

I-I4

**2**

**3**

**4**

**1**

**2**

**3**

**4**

**1**

**2**

**3**

**4**

**1**

**2**

**3**

**4**

**2**

**3**

**4**

**Winery F**

**Winery G**

**Winery H**

**Winery I**

*Restriction patterns obtained with different restriction endonucleases, from the patterns I isolated in four Rioja Alta wineries in year 4.*


#### **Table 7.**

*Clonal diversity of S. cerevisiae yeasts in Rioja Alavesa wineries in 3 consecutive years (1–3).*

exception of the third year of study in winery J. Therefore, the microbiota responsible for the fermentation was different in each harvest.

Regarding the population dynamics of the fermentation, it was observed that in the two wineries, the fermentations were carried out by different *S. cerevisiae* strains that followed each other during the different fermentative phases. In each vinification process, several major clones were detected, which represented at least 30% of the yeasts that carried out the fermentation (**Table 8**). The exception was the third year, in which most of the strains that were found in a fermentative phase were replaced by others in the following stage, particularly in winery K. This fact was also found in some of the vinifications in wineries G and I in Rioja Alta. All four wineries are over a hundred years old and conduct vinifications using the carbonic maceration method.

The comparison of the different strains found in the two cellars during the 3 years of study (**Table 9**) showed the existence of a single common clone, corresponding to the fermentations of the second year. Therefore, the data obtained would show the existence of extensive microbiota in each winery. The fermentations would be the result of the sequence of different populations of yeasts, as there are no representative strains of the sub-zone.

#### *Advances in Grape and Wine Biotechnology*


#### **Table 8.**

*Major S. cerevisiae strains in the fermentations of Rioja Alavesa in 3 consecutive years (1–3).*


*Clones In bold in different columns show the same clone in different cellars*

#### **Table 9.**

*S. cerevisiae clones identified in each elaboration of Rioja Alavesa in 3 consecutive years (1–3).*
