**1. Introduction**

Agricultural residues from food industries are an important raw material involved in bioethanol production. Traditionally, residual juice, molasses and pomace from sugarcane, agave and sugar beet have been widely used in South America for obtaining distilled beverages such us cachaça, tequila and rum. The distillation process is used to isolate, select and concentrate pleasant volatile compounds from the previously fermented liquids and concentrate the alcohol content. Additionally, certain long esters from yeast cells are extracted by distillation and transferred to the final product [1].

Microbial communities from these raw materials and their fermented and distillate beverages not only are interesting due to their role in the aroma production, but their biodiversity and other biotechnological properties are also important. Yeast populations from these ecosystems have not been studied very much, and any studies on them have normally been focused on tequila [2–5], rum [6] and cachaça beverages [7–10].

Yeasts are able to spread from diverse niches to many environments, especially in the vegetable world [11]. Crops and processing plants provide a good niche for yeast growth. In fact, grape crops, musts and wines have been thoroughly studied [12–14], although distillate products and their industry have not been analyzed in Spain in spite of the fact that it is believed to be a new environment for yeast biodiversity study and its biotechnological applications.

In recent years, Spain has been established as the vineyard of the world, presenting the largest surface area (13%) dedicated to this crop [15]. The wine industry is an important sector in Spain which grew considerably throughout 2018. The number of cellars has increased by 6.8% with wine production also increasing (26%) and current production being 40.9 million hectolitres.

Castilla-La Mancha is the world's largest vine-growing region with an annual wine production of around 17 million hectolitres in the 2017–2018 vintage, which accounted for nearly 50% of the total Spanish production. Part of this large production is derived from the distillery industry; in the last year, nearly 250,000 hectolitres were transformed into alcoholic derivatives (16). There is a total of 33 authorized distilleries for wine by-product distillation, 13 of which are located in the La Mancha region. These industries process not only wine but also sweet grape pomaces and its fermented products, obtaining around 4–4.5 million hectolitres [16].

Wine production generates around 600,000 tons of grape derivatives annually such as fermented red skins, which still contain reducing sugars and ethanol, and sweet pomace (from white wine vinification). These by-products, as well as yeast lees and flocculated yeasts, are transported to distilleries where the ethanol is extracted. As **Figure 1** shows, sweet pomaces are mixed and stored for 10–15 days, starting a spontaneous fermentation process. Then, pomace and grape skins are washed with water at 50°C in a heat diffusion system in order to extract the residual sugars and ethanol. After that, a liquid is obtained which is a mixture of alcohol (3–4%) (V/V), water and sugar and is called fermented or sweet piquette. On the other hand, a liquid is drained during the storage of solid organic waste which is mixed with the piquette and fermented for 2–3 days in a stainless-steel container, obtaining a higher alcohol

**167**

**Figure 2.**

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

not been widely studied [19].

**2. Sample collection**

with the aim of extracting the residual ethanol [17, 18].

content (4–5% V/V). Finally, red fermented skins are washed at a lower temperature

The fermented piquettes and the drained liquid are distilled, producing a 93% (V/V) alcohol content product. Then, a dehydration process is carried out until the ethanol concentration of 99.9% is reached. This is mainly used in gasoline as an anti-detonating additive. Residues from distillation can be used as solid fuel (solid

Spontaneous fermentations during this last process are produced by non-*Saccharomyces* and *Saccharomyces* biota present in the environment whose biodiversity has

The lack of information about yeast ecology in this habitat and, more specifi-

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

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

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

residue or "bagasse") or as fertilizer (liquid residue or "vinasse") [17].

cally, in this territory has prompted the aim of this research.

the production of bioethanol and distillates in Spain.

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

under aseptic and refrigerated conditions.

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

*Advances in Grape and Wine Biotechnology*

hectolitres [16].

biodiversity study and its biotechnological applications.

and current production being 40.9 million hectolitres.

Yeasts are able to spread from diverse niches to many environments, especially in the vegetable world [11]. Crops and processing plants provide a good niche for yeast growth. In fact, grape crops, musts and wines have been thoroughly studied [12–14], although distillate products and their industry have not been analyzed in Spain in spite of the fact that it is believed to be a new environment for yeast

In recent years, Spain has been established as the vineyard of the world, presenting the largest surface area (13%) dedicated to this crop [15]. The wine industry is an important sector in Spain which grew considerably throughout 2018. The number of cellars has increased by 6.8% with wine production also increasing (26%)

Castilla-La Mancha is the world's largest vine-growing region with an annual

Wine production generates around 600,000 tons of grape derivatives annually such as fermented red skins, which still contain reducing sugars and ethanol, and sweet pomace (from white wine vinification). These by-products, as well as yeast lees and flocculated yeasts, are transported to distilleries where the ethanol is extracted. As **Figure 1** shows, sweet pomaces are mixed and stored for 10–15 days, starting a spontaneous fermentation process. Then, pomace and grape skins are washed with water at 50°C in a heat diffusion system in order to extract the residual sugars and ethanol. After that, a liquid is obtained which is a mixture of alcohol (3–4%) (V/V), water and sugar and is called fermented or sweet piquette. On the other hand, a liquid is drained during the storage of solid organic waste which is mixed with the piquette and fermented for 2–3 days in a stainless-steel container, obtaining a higher alcohol

*Flowchart that shows all the steps involved in alcohol production from skin, lees and pomace.*

wine production of around 17 million hectolitres in the 2017–2018 vintage, which accounted for nearly 50% of the total Spanish production. Part of this large production is derived from the distillery industry; in the last year, nearly 250,000 hectolitres were transformed into alcoholic derivatives (16). There is a total of 33 authorized distilleries for wine by-product distillation, 13 of which are located in the La Mancha region. These industries process not only wine but also sweet grape pomaces and its fermented products, obtaining around 4–4.5 million

**166**

**Figure 1.**

content (4–5% V/V). Finally, red fermented skins are washed at a lower temperature with the aim of extracting the residual ethanol [17, 18].

The fermented piquettes and the drained liquid are distilled, producing a 93% (V/V) alcohol content product. Then, a dehydration process is carried out until the ethanol concentration of 99.9% is reached. This is mainly used in gasoline as an anti-detonating additive. Residues from distillation can be used as solid fuel (solid residue or "bagasse") or as fertilizer (liquid residue or "vinasse") [17].

Spontaneous fermentations during this last process are produced by non-*Saccharomyces* and *Saccharomyces* biota present in the environment whose biodiversity has not been widely studied [19].

The lack of information about yeast ecology in this habitat and, more specifically, in this territory has prompted the aim of this research.
