**2. Sample collection**

Six of the largest distilleries in Europe, which are at least 100 years old and are found in the towns of Argamasilla de Alba (A), Campo de Criptana (B), Madridejos (C), Villarrobledo (D), Daimiel (E) and Tomelloso (F) in the La Mancha region (**Figure 2**), were selected to carry out the study. La Mancha is the principal area for the production of bioethanol and distillates in Spain.

A total of 47 samples were randomly collected from sweet piquettes [20], fermented piquettes [19], flocculated lees [7] and plant oil [1] throughout the pomacebased ethanol production process, and they were transported to the laboratory under aseptic and refrigerated conditions.

Samples and/or their dilutions were spread on YPD agar plates (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 20 g/L agar); chloramphenicol and sodium propionate were added to inhibit bacteria and mold growth, respectively. Plates were incubated at 28°C/72 hours. Then, samples displaying fewer than 30 colonies were centrifuged to concentrate the cells, and the pellet was directly spread

**Figure 2.** *Location of the distilleries included in this research in the La Mancha region (Spain).*

on YPD agar. Plates with sufficiently separated colonies were replicated onto lysine agar medium (Oxoid, Basingstoke, UK) to distinguish between *Saccharomyces* sp. and non-*Saccharomyces* sp.

The isolates were obtained from 19 samples. A sample was not taken from distillery F, which is possibly due to the hot washing of the skins which would drastically decrease the number of cells.

A total of 210 purified isolates were obtained, 144 *Saccharomyces* and 66 non-*Saccharomyces*, and were stored in 15% glycerol at −80°C until they were studied.

### **3. Yeast classification by genetic identification**

*Saccharomyces* spp. yeasts were the predominant profile in all distillery plants. However, the number of non-*Saccharomyces* species varied between distilleries.

#### **3.1 Non-***Saccharomyces* **yeasts**

Genetic species identification was done using the polymerase chain reaction/ restriction fragment length polymorphism (PCR-RFLP) technique, by amplifying the 5.8S rRNA gene using ITS1 and ITS4 [20]. Amplified products were digested (37°C for 7 h) with the three restriction endonucleases *Hinf* I, *Hae* III and *Cfo* I.

Both PCR products and their restriction fragments were separated on agarose gel with GelGreen™ (Biotium), and the results were visualized using a GeneFlash documentation system. For those isolates that could not be identified by PCR-RFLP analysis, the region D1/D2 from the domain 26D rRNA gene was sequenced using NL1 and NL4 primers. If any variation existed due to the action of the NL4 primer, LR6, NL3A and NL2A primers were used as alternatives. Finally, for those samples in which the percentage of identity at species level was less than 99%, the ITS region was sequenced using ITS1 and ITS4 primers [17]. In **Table 1** all isolates are shown, classified at the species level with 99% similarity and the NCBI accession number obtained. A percentage of similarity lower than 99% was obtained with isolates 23, 33, 48 and 62 using the primers NL1/NL4. Sequencing of the 5.8S rRNA + ITS region confirmed this with a similarity of 99%.

Non-*Saccharomyces* yeasts were mainly distributed in sweet piquettes (45.5%) without ethanol, 43.3% were found in fermented piquettes, where the ethanol concentration varied between 4% and 5% (v/v). Finally, 18.2% and 3% were isolated from plant soil and sedimented yeast lees, respectively (**Figure 3**).

As can be observed in **Figure 4**, non-*Saccharomyces* yeasts were more present in plant C (14%) and in plant D (47%) due to the difference in the age of the distilleries and the specific elaboration process followed.

The 66 isolates were cataloged as 8 genera and 20 species, which belonged mainly to the genera *Pichia* (38.0%), *Candida* (22.7%), *Hanseniaspora* (18.2%) and *Torulaspora* (10.6%). The remaining 10% belonged to *Zygosaccharomyces*, *Lachancea*, *Ogataea* and *Saccharomycodes*.

There were four predominant species that were identified as *Pichia galeiformis*, *Torulaspora delbrueckii*, *Hanseniaspora osmophila* and *Candida lactis-condensi*. All these results showed that a considerable diversity exists in this environment, unlike in grape must fermentations [21].

With regard to the substrata of isolation (**Figure 3**), *T. delbrueckii*, *H. osmophila*, *P. kudriavzevii*, *C. lactis-condensi* and *P. anomala* were isolated from sweet piquettes, while *P. galeiformis* and *C. ethanolica* were found in fermented piquettes, from which other species, such us *S'codes ludwigii*, *P. bimundalis*, *Zygosaccharomyces bailii* and *C. sake*, were also isolated but at a very low percentage. Only two species, *L. thermotolerans* and

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*Yeast from Distillery Plants: A New Approach DOI: http://dx.doi.org/10.5772/intechopen.86291*

*Candida ethanolica* 35a, 36c, 40b

*Candida lactis-condensi* 50c, 51c, 52c

*Hanseniaspora osmophila* 4b, 26a, 58a

*Pichia anomala* 10c, 20a, 21a,

*Pichia galeiformis* 9c, 37a, 38a,

*Pichia kudriavzevii* 3 a, b, 8a, 13a,

**Species Isolates Nº Accession number (NCBI)**

41a, 48a,

53c, 54c, 55a 56a, 57a

59a, 62 a, d, 65a, 66a

*Candida sake* 44a JX880410 *Candida viswanathii* 39a JQ512833 *Hanseniaspora meyeri* 7a JN248602

*Hanseniaspora uvarum* 11a, 28 a, d 11/JN248600

*Hanseniaspora valbyensis* 5a JN248613 *Hanseniaspora vineae* 2a JN248606 *Lachancea thermotolerans* 25 a, b, 46a 25/JQ073770

*Ogataea polymorpha* 19a JN248599

*Pichia bimundalis* 43b JQ073768

*Pichia membranaefaciens* 23 a, d 23a/JQ410476

22a, 27a, 32a

45b, 47c, 49c, 68c, 69c, 70c, 71c, 74b, 76c

14b, 24b

35/JX880409 40/JQ073769 41/JX880400 48/JQ410478

55/JN248610 56/JN248614 57/JN248611

4/JQ073772 26/JQ512831 59/JQ512840 58/JQ512840 62a/JQ410479 62d/JQ410479 65/JQ512841 66/JQ780464

28/JN512834-9

46/JN248601

20/JX880399 21/JX880404 22/JN248608 27/JX880405 32/JX880406

37/JX880397 38/JX880398 45/JQ073767 74/JQ073765

3/JN248607 8/JN248609 13/JX880402 14/JQ073771 24/JQ073766

23d/JQ410476

### *Yeast from Distillery Plants: A New Approach DOI: http://dx.doi.org/10.5772/intechopen.86291*

*Advances in Grape and Wine Biotechnology*

and non-*Saccharomyces* sp.

decrease the number of cells.

**3.1 Non-***Saccharomyces* **yeasts**

**3. Yeast classification by genetic identification**

region confirmed this with a similarity of 99%.

eries and the specific elaboration process followed.

*Ogataea* and *Saccharomycodes*.

in grape must fermentations [21].

on YPD agar. Plates with sufficiently separated colonies were replicated onto lysine agar medium (Oxoid, Basingstoke, UK) to distinguish between *Saccharomyces* sp.

A total of 210 purified isolates were obtained, 144 *Saccharomyces* and 66 non-*Saccharomyces*, and were stored in 15% glycerol at −80°C until they were studied.

*Saccharomyces* spp. yeasts were the predominant profile in all distillery plants. However, the number of non-*Saccharomyces* species varied between distilleries.

Genetic species identification was done using the polymerase chain reaction/ restriction fragment length polymorphism (PCR-RFLP) technique, by amplifying the 5.8S rRNA gene using ITS1 and ITS4 [20]. Amplified products were digested (37°C for 7 h) with the three restriction endonucleases *Hinf* I, *Hae* III and *Cfo* I. Both PCR products and their restriction fragments were separated on agarose gel with GelGreen™ (Biotium), and the results were visualized using a GeneFlash documentation system. For those isolates that could not be identified by PCR-RFLP analysis, the region D1/D2 from the domain 26D rRNA gene was sequenced using NL1 and NL4 primers. If any variation existed due to the action of the NL4 primer, LR6, NL3A and NL2A primers were used as alternatives. Finally, for those samples in which the percentage of identity at species level was less than 99%, the ITS region was sequenced using ITS1 and ITS4 primers [17]. In **Table 1** all isolates are shown, classified at the species level with 99% similarity and the NCBI accession number obtained. A percentage of similarity lower than 99% was obtained with isolates 23, 33, 48 and 62 using the primers NL1/NL4. Sequencing of the 5.8S rRNA + ITS

Non-*Saccharomyces* yeasts were mainly distributed in sweet piquettes (45.5%) without ethanol, 43.3% were found in fermented piquettes, where the ethanol concentration varied between 4% and 5% (v/v). Finally, 18.2% and 3% were isolated

As can be observed in **Figure 4**, non-*Saccharomyces* yeasts were more present in plant C (14%) and in plant D (47%) due to the difference in the age of the distill-

There were four predominant species that were identified as *Pichia galeiformis*, *Torulaspora delbrueckii*, *Hanseniaspora osmophila* and *Candida lactis-condensi*. All these results showed that a considerable diversity exists in this environment, unlike

With regard to the substrata of isolation (**Figure 3**), *T. delbrueckii*, *H. osmophila*, *P. kudriavzevii*, *C. lactis-condensi* and *P. anomala* were isolated from sweet piquettes, while *P. galeiformis* and *C. ethanolica* were found in fermented piquettes, from which other species, such us *S'codes ludwigii*, *P. bimundalis*, *Zygosaccharomyces bailii* and *C. sake*, were also isolated but at a very low percentage. Only two species, *L. thermotolerans* and

The 66 isolates were cataloged as 8 genera and 20 species, which belonged mainly to the genera *Pichia* (38.0%), *Candida* (22.7%), *Hanseniaspora* (18.2%) and *Torulaspora* (10.6%). The remaining 10% belonged to *Zygosaccharomyces*, *Lachancea*,

from plant soil and sedimented yeast lees, respectively (**Figure 3**).

The isolates were obtained from 19 samples. A sample was not taken from distillery F, which is possibly due to the hot washing of the skins which would drastically

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*Technique that allowed identification: (a) NL1/NL4 primers; (b) NL2A/LR6 and NL2A/NL3A primers; (c) PCR-RFLP; (d) 1.8S–5.8S rRNA region sequence (ITS1/ITS4 primers).*

#### **Table 1.**

*Yeast isolates identified in the different distilleries studied and accession number (NCBI).*

#### **Figure 3.**

*Percentage of yeast species isolated in sweet and fermented piquettes, lees and plant soil.*

*T. delbrueckii*, were found equally frequently in both sweet and fermented piquettes. Having analysed all these results, a large biodiversity of yeasts was found in the studied substrata, as was documented for grape marc by Bovo et al. [22, 23].

On the other hand, the distribution of genera (**Figure 4a**) and species (**Figure 4b**) in the studied distilleries was also analysed. *Candida* and *Pichia* genera were found in almost all of them, and *Torulaspora* and *Hanseniaspora* were found in three of the five plants in which yeasts were isolated. *P. galeiformis*, *P. kudriavzevii*, *T. delbrueckii* and *H. osmophila* were the species identified in most of the ethanol plants, with plant A being the only one where no major species were found, which contrasts with the results for the other plants (**Figure 4b**).

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**Figure 4.**

The presence of *Candida* species (*C. sake*, *C. sorbosa*, *C. stellata*, *C. guilliermon-*

*K. marxianus* and large *Saccharomyces* spp. populations has been previously documented in Brazilian distilleries [7, 8]. These results confirmed that the yeast profiles in the distilleries of the two regions are very different and it is evident that the

*dii*, *C. karawaiewii* and *C. citrea*), *P. membranaefaciens*, *P. guilliermondii*,

*Distribution of genus (4a) and species (4b) in distilleries studied.*

*Yeast from Distillery Plants: A New Approach DOI: http://dx.doi.org/10.5772/intechopen.86291* *Yeast from Distillery Plants: A New Approach DOI: http://dx.doi.org/10.5772/intechopen.86291*

*Advances in Grape and Wine Biotechnology*

*RFLP; (d) 1.8S–5.8S rRNA region sequence (ITS1/ITS4 primers).*

**170**

**Figure 3.**

**Table 1.**

*T. delbrueckii*, were found equally frequently in both sweet and fermented piquettes. Having analysed all these results, a large biodiversity of yeasts was found in the studied

On the other hand, the distribution of genera (**Figure 4a**) and species (**Figure 4b**) in the studied distilleries was also analysed. *Candida* and *Pichia* genera were found in almost all of them, and *Torulaspora* and *Hanseniaspora* were found in three of the five plants in which yeasts were isolated. *P. galeiformis*, *P. kudriavzevii*, *T. delbrueckii* and *H. osmophila* were the species identified in most of the ethanol plants, with plant A being the only one where no major species were found, which contrasts with the

**Species Isolates Nº Accession number (NCBI)** *S'codes ludwigii* 72a, 77a 72/JX880401

*Torulaspora delbrueckii* 1a, 6a, 60a, 61a, 64a, 67a, 75a 1/JN248605

*Zygosaccharomyces bailii* 34a JN248597 *Zygosaccharomyces fermentati* 15a JX880403 *Technique that allowed identification: (a) NL1/NL4 primers; (b) NL2A/LR6 and NL2A/NL3A primers; (c) PCR-*

*Yeast isolates identified in the different distilleries studied and accession number (NCBI).*

77/JQ512842

6/JQ780463 60/JX880407 61/JQ512830 64/JX880408 67/JQ512843 75/JQ780465

substrata, as was documented for grape marc by Bovo et al. [22, 23].

*Percentage of yeast species isolated in sweet and fermented piquettes, lees and plant soil.*

results for the other plants (**Figure 4b**).

#### **Figure 4.**

*Distribution of genus (4a) and species (4b) in distilleries studied.*

The presence of *Candida* species (*C. sake*, *C. sorbosa*, *C. stellata*, *C. guilliermondii*, *C. karawaiewii* and *C. citrea*), *P. membranaefaciens*, *P. guilliermondii*, *K. marxianus* and large *Saccharomyces* spp. populations has been previously documented in Brazilian distilleries [7, 8]. These results confirmed that the yeast profiles in the distilleries of the two regions are very different and it is evident that the

Spanish industry is an interesting yeast niche. Additionally, some of these genera and species were also found by Amaya-Delgado et al. [5] and Lappe-Oliveras et al. [4] in tequila and agave beverages.
