Information

Can yeast in a primary fermentation of wine be used as a source of yeast for new fermentation?


So if I were to pitch bread yeast, and get to primary fermentation, which as I understand it, is the point at which regular cellular respiration can no longer continue due to a lack of oxygen , which is needed for the electron transport chain. Therefore the yeast switch to fermentation, which produces my desired product, ethanol.

But as I understand it, at this state the yeast are no longer reproducing and are in a survival mode.

Therefore if I were to cut a 2$L$ bottle of my primary ferment in half, and then add more sugar and water to the now separate 1$L$ bottles, and oxygen (if possible), up to 2$L$, would I be able to have the same amount of ethanol produced/would fermentation even occur?

I feel there would be diminishing returns even if it was possible, since yeast can only reproduce asexually so many times, which happens during stressful times, which this process might induce?


Yes, it is possible to reuse yeast in both beer and wine fermentation - commercial brewers do it all the time for cost savings and batch reproducibility, and although I'm not as familiar with making wine, many sites including this one say it's perfectly fine, as long as the viability of the cells is high enough.

The yeast aren't necessarily in stress-induced survival mode during fermentation, they're just living (and metabolizing) anaerobically. They may no longer be reproducing, or doing it very infrequently, but they'll remain perfectly happy little buggers (that's a technical term) for quite a while. Give them more food (sugar), and they'll keep fermenting. What eventually stops the process with wine is the level of ethanol rising too high for their comfort. If you were to dilute the ethanol out with water, they'd keep going.

Now, I wouldn't recommend doing this indefinitely (some strains may exhibit genomic instability, etc.), but new batches can certainly be produced with existing pitches.


Fermentation

The alcoholic beverages that can be produced by fermentation vary widely, depending primarily on two factors&mdashthe plant that is fermented and the enzymes used for fermentation. Human societies use, of course, the materials that are available to them. Thus, various peoples have used grapes, berries, corn, rice, wheat, honey, potatoes, barley, hops, cactus juice, cassava roots, and other plant materials for fermentation. The products of such reactions are various forms of beer, wine or distilled liquors, which may be given specific names depending on the source from which they come. In Japan, for example, rice wine is known as sake. Wine prepared from honey is known as mead. Beer is the fermentation product of barley, hops, and/or malt sugar.

Early in human history, people used naturally occurring yeast for fermentation. The products of such reactions depended on whatever enzymes might occur in "wild" yeast. Today, wine-makers are able to select from a variety of specially cultured yeast that control the precise direction that fermentation will take.

Ethyl alcohol is not the only useful product of fermentation. The carbon dioxide generated during fermentation is also an important component of many baked goods. When the batter for bread is mixed, for example, a small amount of sugar and yeast is added. During the rising period, sugar is fermented by enzymes in the yeast, with the formation of carbon dioxide gas. The carbon dioxide gives the batter bulkiness and texture that would be lacking without the fermentation process.

Fermentation has a number of commercial applications beyond those described thus far. Many occur in the food preparation and processing industry. A variety of bacteria are used in the production of olives, cucumber pickles, and sauerkraut from the raw olives, cucumbers, and cabbage, respectively. The selection of exactly the right bacteria and the right conditions (for example, acidity and salt concentration) is an art in producing food products with exactly the desired flavors. An interesting line of research in the food sciences is aimed at the production of edible food products by the fermentation of petroleum.

In some cases, antibiotics and other drugs can be prepared by fermentation if no other commercially efficient method is available. For example, the important drug cortisone can be prepared by the fermentation of a plant steroid known as diosgenin. The enzymes used in the reaction are provided by the mold Rhizopus nigricans.

One of the most successful commercial applications of fermentation has been the production of ethyl alcohol for use in gasohol. Gasohol is a mixture of about 90% gasoline and 10% alcohol. The alcohol needed for this product can be obtained from the fermentation of agricultural and municipal wastes. The use of gasohol provides a promising method for using renewable resources (plant material) to extend the availability of a nonrenewable resource (gasoline).

Another application of the fermentation process is in the treatment of wastewater. In the activated sludge process, aerobic bacteria are used to ferment organic material in wastewater. Solid wastes are converted to carbon dioxide, water, and mineral salts.


Preparing for the fermentation

demijohn with
air lock

Once the grapes have been pressed and the acidity and sugar levels have been checked and adjusted as necessary, it is time to prepare the must for fermentation.

The must should now be in a fermentation vessel of some type - but not filled right to the top. You will then want to add to the must the following ingredients:

  1. Yeast Nutrient: add at the rate of 1 teaspoon per gallon. This is not yeast, but an energy source for the yeast which will be added later.
  2. Pectic Enzyme: add at the rate of 1/8 teaspoon per gallon. This is used to aid in the clarification of the wine, and in the case of red wines, to help break down the pulp so more flavor can be extracted.
  3. Potassium Bisulfite: add at the rate of 1/16 teaspoon per gallon or 1/4 teaspoon for every 4 gallons. This is used to sterilize the must, to kill all the wild molds, bacteria and yeast that come with the fresh grapes. Over a 24 hour period the Potassium Bisulfite will sterilize the juice and then dissipate into the air. Only cover the fermentation vessel with a light towel during the waiting period.
  4. Yeast can then be added after waiting 24 hours. If the yeast is added before the Potassium Bisulfite leaves, it will kill the yeast as well. Just sprinkle the yeast onto the surface of the must at a rate of 1 package for every 5 gallons.

Once the fermentation gets under way, try to ensure that the temperature is cool and stable.

TOO HOT: YEAST MAY OVERHEAT, AND DIE QUICKLY.
TOO COLD: MAY END UP WITH A STUCK FERMENTATION.


Molecular Identification and Characterization of Wine Yeasts

M. Teresa Fernández-Espinar , . Eladio Barrio , in Molecular Wine Microbiology , 2011

4.3 Characterization of Commercial Yeasts

Dried wine yeasts were developed in the 1950s when laboratories in Canada ( Adams, 1954 ) and the United States ( Castor, 1953 ) independently carried out selection of wine strains that were subsequently used in directed fermentations. More than 100 different strains are currently marketed, mainly by six companies. Molecular characterization of commercial yeast strains is necessary for two reasons. Firstly, it is needed for quality-control purposes to confirm that the obtained yeast is the one that was originally selected and not a contaminant, and, secondly, to detect fraud.

Given that most active dried yeasts belong to the species S. cerevisiae, the techniques used must be able to differentiate clearly between strains. Most of the techniques described in Section 3 are useful for this purpose, as was recently shown by Schuller et al. (2004) in a comparative study of 23 commercial strains by electrophoretic karyotyping, restriction analysis of mtDNA, amplification of δ elements, and microsatellite analysis. Electrophoretic karyotyping ( Blondin & Vezinhet, 1998 Yamamoto et al., 1991 ), amplification of δ elements ( Legras & Karst, 2003 Ness et al., 1993 ), and microsatellite analysis ( González Techera et al., 2001 ) had previously been used for this purpose. Other studies have been reported in which more than one technique was used to characterize commercial isolates: mtDNA analysis and karyotyping ( Schuller et al., 2004 Vezinhet et al., 1990 ), amplification of δ elements and DNA fingerprinting ( Lavallée et al., 1994 ), and karyotyping with hybridization ( Degré et al., 1989 ). In fact, Fernández-Espinar et al. (2001) showed that definitive characterization of commercial strains requires a combination of various molecular techniques. The techniques applied in that study were restriction analysis of mtDNA with HinfI, electrophoretic karyotyping, and PCR amplification of genomic δ elements. One of the most interesting findings reported by Fernández-Espinar et al. (2001) was the large number of errors or fraudulent practices by companies that produce commercial yeasts. Commercial strains have also been characterized by Echeverrigaray et al. (2000) using the RAPD technique and by Manzano et al. (2006) using TGGE-PCR and restriction analysis. De Barros Lopes et al. (1996) have developed a technique based on amplification of introns for the characterization of commercial strains. However, this technique has not been applied subsequently by other authors, possibly as a result of the complexity of the profiles generated.


Making the Wine

To vinify is to turn grape juice into wine. To extract the juice, grapes are pressed using a wine press. The freshly pressed grape juice that contains the skins, seeds, and stems of the fruit is called must everything but the juice (skins, seeds, etc.) is called pomace or marc.

Primary fermentation is the initial fermentation, in which yeast convert sugars in grape juice or must to alcohol (wine) and carbon dioxide. Yeasts are single-celled microorganism that convert sugar to alcohol and carbon dioxide during the fermentation stage of wine production. During malolactic fermentation, which occurs after primary fermentation, tart malic acid, which occurs naturally in grape must, is converted to milder, softer-tasting lactic acid. Secondary fermentation is either a continuation of the primary fermentation of sugar to alcohol that takes place after the wine is moved from one type of container to another, such as from stainless steel to oak, or a supplemental fermentation triggered after the primary fermentation is complete by the addition of sugars, such as is commonly done in the production of sparkling wines. This differs from chaptalizing, or adding sugar to the must or juice before fermentation to make up for deficiencies in vine-ripened sugar levels.

At any point during the winemaking process, a hydrometer may be used to measure sugar content (brix). A hydrometer is a calibrated glass float used to measure the specific gravity (relative density) of liquids.

Topping up involves replacing wine that has evaporated from a barrel, ensuring no air space in the container and no exposure to oxygen. Sulfites are sulfur-based compounds that occur naturally during wine fermentation, but are also often added before, during, or after fermentation.They protect wine from oxidation and the acitivity of undesirable microorganisms, particularly bacteria. Sulfites are typically added at higher levels to white and/or sweet wines to prevent browning and/or spoilage. Wines may be blended, or mixed. Often, a blending cultivar is grown specifically to be mixed with other grapes in the winery.


The Chemistry of Wine: Fermentation

There are two basic ingredients needed to ferment the juice of grapes into wine: sugar and yeast. Like all fruit, sugar is found naturally in grapes, with the sugar level increasing as the grapes ripen on the vine a process in the wine-making world called veraison. Ripening can take one to two months, depending on the climate. The right balance of rain and sunshine ensures good sugar levels in the grapes. When ready, the grapes are picked and crushed, leaving the juice, known as must, for fermentation.

The second ingredient needed for fermentation, yeast, consumes the sugar in the must, and as a byproduct, it releases three components: ethanol, CO2, and heat. The CO2 and heat escape, and the ethanol remains.

The yeast needed for fermentation can be found naturally in the environment and on the grapes themselves. This natural yeast dies off, however, when grape juice reaches 4 to 5 percent alcohol by volume, before fermentation is complete.

In order to ferment the must completely, then, the winemaker adds an anaerobic (no oxygen needed), cultured yeast called Saccharomyces cerevisiae. Depending on the temperature at which must is fermented, the process can take one to two weeks.

Following fermentation, the winemaker will store the wine in various vessels, such as barrels or stainless-steel tanks, for example, for a period of time designated by local wine laws and based on the style of wine being made. During that period, harsh acids in the wine convert into softer, more palatable acids. (Some grape varieties might need a little help, so the winemaker will kickstart the process.) Once bottled, the wine may be stored for even longer to age before reaching your table.

Whether you fancy red, white, sparkling or fortified wine, fermentation is the chemical reaction at the heart of the process. It is a practice that has been honed over thousands of years, spreading around the world and surviving history to the modern-day wine-making that we enjoy today.


Contents

For most of the history of wine, winemakers did not know the mechanism that somehow converted sugary grape juice into alcoholic wine. They could observe the fermentation process which was often described as "boiling", "seething" or the wine being "troubled" due to release of carbon dioxide that gave the wine a frothy, bubbling appearance. This history is preserved in the etymology of the word "yeast" itself which essentially means "to boil". [3] [6]

In the mid-19th century, the French scientist Louis Pasteur was tasked by the French government to study what made some wines spoil. His work, which would later lead to Pasteur being considered one of the "Fathers of Microbiology", would uncover the connection between microscopic yeast cells and the process of the fermentation. It was Pasteur who discovered that yeast converted sugars in the must into alcohol and carbon dioxide, though the exact mechanisms of how the yeast would accomplish this task was not discovered till the 20th century with the Embden–Meyerhof–Parnas pathway. [7]

The yeast species commonly known as Saccharomyces cerevisiae was first identified in late 19th century enology text as Saccharomyces ellipsoideus due to the elliptical (as opposed to circular) shape of the cells. Throughout the 20th century, more than 700 different strains of Saccharomyces cerevisiae were identified. The difference between the vast majority of these strains are mostly minor, though individual winemakers will develop a preference for particular strains when making certain wines or working with particular grape varieties. Some of these difference include the "vigor" or speed of fermentation, temperature tolerance, the production of volatile sulfur compounds (such as hydrogen sulfide) and other compounds that may influence the aroma of the wine. [3]

The primary role of yeast is to convert the sugars present (namely glucose) in the grape must into alcohol. The yeast accomplishes this by utilizing glucose through a series of metabolic pathways that, in the presence of oxygen, produces not only large amounts of energy for the cell but also many different intermediates that the cell needs to function. In the absence of oxygen (and sometimes even in the presence of oxygen [8] ), the cell will continue some metabolic functions (such as glycolysis) but will rely on other pathways such as reduction of acetaldehyde into ethanol (fermentation) to "recharge" the co-enzymes needed to keep metabolism going. It is through this process of fermentation that ethanol is released by the yeast cells as a waste product. Eventually, if the yeast cells are healthy and fermentation is allowed to run to the completion, all fermentable sugars will be used up by the yeast with only the unfermentable pentose leaving behind a negligible amount of residual sugar. [4]

Other compounds in wine produced by yeast Edit

While the production of alcohol is the most noteworthy by-product of yeast metabolism from a winemaking perspective, there are a number of other products that yeast produce that can be also influence the resulting wine. This includes glycerol which is produced when an intermediate of the glycolysis cycle (dihydroxyacetone) is reduced to "recharge" the NADH enzyme needed to continue other metabolic activities. [4] This is usually produced early in the fermentation process before the mechanisms to reduce acetaldehyde into ethanol to recharge NADH becomes the cell's primary means of maintaining redox balance. As glycerol contributes increased body and a slightly sweet taste without increasing the alcohol level of the wine, some winemakers try to intentionally favor conditions that would promote glycerol production in wine. This includes selecting yeast strains that favor glycerol production (or allowing some wild yeast like Kloeckera and Metschnikowia to ferment), increased oxygen exposure and aeration as well as fermenting at higher temperatures. [8] Glycerol production is also encouraged if most available acetaldehyde is made unavailable by binding with bisulfite molecules in the wine, but it would take a substantial amount of sulfur dioxide addition (far beyond legal limits) to prolong glycerol production beyond just these very nascent stages of fermentation. [9]

Other by-products of yeast include: [3] [9]

    – Caused by the demethylation of pectins in the must by enzymes of the yeast. More commonly found in red wines than white but only in very small amounts between 20–200 mg/L. – Formed by the decomposition of amino acids by the yeast. This includes 2,3-Butanediol which is formed by yeast-consuming diacetyl, the compound that gives Chardonnay and other wines a "buttery" aroma, reducing it first to acetoin and then to the more neutral-smelling 2,3-Butanediol. Many beer and winemakers who have a wine with too much "butteriness" will often "pitch" fresh yeast cultures into the no longer fermenting tank so that the yeast will consume the diacetyl and reduce the aroma. [10] – Like glycerol, this is often formed early in fermentation. Usually found in concentrations of 500–1200 mg/L, it is a minor acid in the overall acidity of wine. – Considered a main component of volatile acidity that can make a wine taste unbalanced and overly acidic. While acetic acid is the main volatile acid produced by yeast, trace amounts of butyric, formic and propionic acids can also be formed depending on the yeast strain. Most countries have wine laws setting the legal limit of volatile acidity, usually expressed as acetic acid, to 1200–2000 mg/L. Acetic acid can also lead to the development of the wine fault ethyl acetate which is characterized by a "nail polish remover" smell. However, small amounts of acetic acid are actually beneficial for the yeast as they use them to synthesis lipids in the cell membrane. [3]
    – While most of the acetaldehyde produce gets reduced to ethanol or is bound by sulfur dioxide, concentrations between 50–100 mg/L can remain in the wine. The flor yeast strains that produce the Spanish wineSherry will produce higher amounts that contributes to the characterized "aldehydic" aromas of Sherries. In the presence of oxygen, yeast can convert some of the ethanol presence in the wine back into acetaldehyde creating oxidized aromas. [3] – Often produced by yeast during fermentation because of a nitrogen deficiency in the must. This can be done by a reduction of sulfates or sulfites available in the must or by the decomposition of dead yeast cells by other yeast that releases sulfur-containing amino acids that are further broken down by the yeast. The latter often happens with wines that sit in contact with their lees for long periods of time between rackings. In the presence of alcohol, hydrogen sulfide can react with ethanol to form ethyl mercaptans and disulfides that contribute to off aromas and wine faults. Some commercial yeast strains, such as Montrachet 522 are known to produce higher levels of hydrogen sulfides than other strains, particularly if the must has some nutrient deficiencies. [1] – Along with acetaldehyde, this compound can react with anthocyanins extracted from contact with grape skins to create a more stable color pigment (pyranoanthocyanin) that can enhance the color of some red wines. [3]
  • Various esters, ketones, lactones, phenols and acetals. [2]

Lees Edit

When yeast cells die, they sink to the bottom of the fermentation vessel where they combine with insoluble tartrates, grape seeds, skin and pulp fragments to form the lees. During fermentation, the first significant racking which removes the bulk of dead yeast cells is often referred to as the gross lees as opposed to the less coarse fine lees that come as the wine continues to settle and age. During the time that the wine spends in contact with the lees, a number of changes can impact the wine due to both the autolysis (or self-metabolize) of the dead yeast cells as well as the reductive conditions that can develop if the lees are not aerated or stirred (a process that the French call bâtonnage). The length of time that a wine spends on its lees (called sur lie) will depend on the winemaking style and type of wine. [11]

The process of leaving the wine to spend some contact with the lees has a long history in winemaking, being known to the Ancient Romans and described by Cato the Elder in the 2nd century BC. Today the practice is widely associated with any red wines that are barrel fermented, Muscadet, sparkling wine Champagne as well as Chardonnay produced in many wine regions across the globe. Typically when wines are left in contact with their lees, they are regularly stirred in order to release the mannoproteins, polysaccharides and other compounds that were present in the yeast cell walls and membranes. This stirring also helps avoid the development of reductive sulfur compounds like mercaptans and hydrogen sulfide that can appear if the lees layer is more than 10 cm (4 inches) thick and undisturbed for more than a week. [11]

Most of the benefits associated with lees contact deals with the influence on the wine of the mannoproteins released during the autolysis of the yeast cells. Composed primarily of mannose and proteins, with some glucose, mannoproteins are often bound in the cell wall of yeast with hydrophobic aroma compounds that become volatilized as the cell wall breaks down. Not only does the release of mannoproteins impart sensory changes in the wine but they can contribute to tartrate and protein stability, help enhance the body and mouthfeel of the wine as well as decrease the perception of bitterness and astringency of tannins. [4]

Secondary fermentation Edit

The production of Champagne and many sparkling wines requires a second fermentation to occur in the bottle in order to produce the carbonation necessary for the style. A small amount of sugared liquid is added to individual bottles, and the yeast is allowed to convert this to more alcohol and carbon dioxide. The lees are then ricked into the neck of the bottle, frozen, and expelled via pressure of the carbonated wine.

Yeast taxonomy includes classification of yeast species depending on the presence or absence of a sexual phase. Therefore, some winemaking yeasts are classified by their asexual anamorph (or "imperfect" form) while others may be classified by their sexual teleomorph (or "perfect" form). A common example of this is Brettanomyces (or "Brett") that is usually referenced in wine and viticulture text under its asexual classification though some scientific and winemaking texts may describe specific species (such as Dekkera bruxellensis) under its sporulating sexual classification of Dekkera. [4] Unless otherwise noted, this article will commonly refer to the asexual form of wine yeast.

The most common yeast generally associated with winemaking is Saccharomyces cerevisiae which is also used in bread making and brewing. Other genera of yeast that can be involved in winemaking (either beneficially or as the cause of potential wine faults) include: [3] [4]

  • Brettanomyces (Teleomorph Dekkera)
  • Candida (Teleomorphs for different species from several genera including Pichia, Metschnikowia, Issatchenkia, Torulaspora and Kluyveromyces)
  • Kloeckera (Teleomorph Hanseniaspora), usually the most common "wild yeast" found in the vineyard. Some species are known as "killer yeast" that produce inhibitory levels of ethyl acetate and acetic acid that can kill off sensitive strains of Saccharomyces cerevisiae[5]
  • Saccharomycodes
  • Schizosaccharomyces, the only wine yeast that reproduced by fission whereas most wine yeast reproduce by budding. [4]
  • Zygosaccharomyces, very alcohol-tolerant and can grow in wines up to 18% v/v. Additionally this yeast can survive in extremely high sugar levels (as much as 60% w/w or 60 Brix) and is very resistant to sulfur dioxide. [4]
  • Aureobasidium, particularly the "black yeast" species of Aureobasidium pullulans found in moist cellars that can contaminate aging wine in barrels. [4]

Saccharomyces Edit

The yeast genus Saccharomyces (sugar mold) is favored for winemaking (for both grapes as well as other fruit wines in addition also to being used in brewing and breadmaking) because of the generally reliable and positive attributes it can bring to the wine. These yeasts will usually readily ferment glucose, sucrose and raffinose and metabolize glucose, sucrose, raffinose, maltose and ethanol. However, Saccharomyces cannot ferment or utilize pentoses (such as arabinose) which is usually present in small amount in wines as residual sugars. [4]

In addition to Saccharomyces cerevisiae, other species within the genus Saccharomyces that are involved with winemaking include: [1] [3] [4]

Influences of different strains on fermentation Edit

In 1996, Saccharomyces cerevisiae was the first single-celled, eukaryotic organism to have its entire genome sequenced. This sequencing helped confirm the nearly century of work by mycologists and enologists in identifying different strains of Saccharomyces cerevisiae that are used in beer, bread and winemaking. Today there are several hundred different strains of S. cerevisiae identified. [3] Not all of the strains are suitable for winemaking and even among the strains that are, there is debate among winemakers and scientists about the actual magnitude of differences between the various strains and their potential impact on the wine. [5] Even among strains that have demonstrated distinctive difference when compared among young wines, these differences seem to fade and become less distinctive as the wines age. [2]

Some distinct difference among various strains include the production of certain "off-flavor" and aromas that may be temporary (but producing a "stinky fermentation") or could stay with the wine and either have to be dealt with through other winemaking means (such as the presence of volatile sulfur compounds like hydrogen sulfide) or leave a faulty wine. Another difference includes the "vigor" or speed of fermentation (which can also be influenced by other factors beyond yeast selection) with some yeast strains having the tendency to do "fast ferments" while others may take longer to get going. [3]

Another less measurable difference that are subject to more debate and questions of winemakers preference is the influence of strain selection on the varietal flavors of certainly grape varieties such as Sauvignon blanc and Sémillon. It is believed that these wines can be influenced by thiols produced by the hydrolysis of certain cysteine-linked compounds by enzymes that are more prevalent in particular strains. Other aromatic varieties such as Gewürztraminer, Riesling and Muscat may also be influenced by yeast strains containing high levels of glycosidases enzymes that can modify monoterpenes. Similarly, though potentially to a much smaller extent, other varieties could be influenced by hydrolytic enzymes working on aliphatics, norisoprenoids, and benzene derivatives such as polyphenols in the must. [3]

In sparkling wine production some winemakers select strains (such as one known as Épernay named after the town in the Champagne wine region of France and California Champagne, also known as UC-Davis strain 505) that are known to flocculate well, allowing the dead yeast cells to be removed easily by riddling and disgorgement. In Sherry production, the surface film of yeast known as flor used to make the distinctive style of fino and manzanilla sherries comes from different strains of Saccharomyces cerevisiae, [3] though the commercial flor yeast available for inoculation is often from different species of Saccharomyces, Saccharomyces beticus, Saccharomyces fermentati and Saccharomyces bayanus. [1] [2] [5]

In winemaking, the term "wild yeast" has multiple meanings. In its most basic context, it refers to yeast that has not been introduced to the must by intentional inoculation of a cultured strain. Instead, these "wild yeasts" often come into contact with the must through their presence on harvest equipment, transport bins, the surface winemaking equipment and as part of the natural flora of a winery. Very often these are strains of Saccharomyces cerevisiae that have taken residence in these places over the years, sometimes being previously introduced by inoculation of prior vintages. In this context, these wild yeasts are often referred to as ambient, indigenous or natural yeast as opposed to inoculated, selected or cultured yeast. Wineries that often solely rely on these "in-house" strains will sometimes market their wines as being the product of wild or natural fermentations. [3] The (c. 304) Nanfang Caomu Zhuang has the earliest description of winemaking using "herb ferment" (cǎoqū 草麴) wild yeast with rice and various herbs, including the poisonous Gelsemium elegans (yěgé 冶葛). [12] [13]

Another use of the term "wild yeast" refers to the non-Saccharomyces genera of yeasts that are present in the vineyard, on the surface of grapevines and of the grapes themselves. Anywhere from 160 to 100,000 colony forming units of wild yeasts per berry could exist in a typical vineyard. These yeasts can be carried by air currents, birds and insects through the vineyard and even into the winery (such as by fruit flies). The most common wild yeasts found in the vineyard are from the genera Kloeckera, Candida and Pichia with the species Kloeckera apiculata being the most dominant species by far. [5] Saccharomyces cerevisiae, itself, is actually quite rarely found in the vineyard or on the surface freshly harvested wine grapes unless the winery frequently reintroduced winery waste (such as lees and pomace) into the vineyard. [3]

Unlike the "ambient" Saccharomyces wild yeast, these genera of wild yeasts have very low tolerance to both alcohol and sulfur dioxide. They are capable of starting a fermentation and often begin this process as early as the harvest bin when clusters of grapes get slightly crushed under their own weight. Some winemakers will try to "knock out" these yeasts with doses of sulfur dioxide, most often at the crusher before the grapes are pressed or allowed to macerate with skin contact. Other winemakers may allow the wild yeasts to continue fermenting until they succumb to the toxicity of the alcohol they produce which is often between 3–5% alcohol by volume and then letting either inoculated or "ambient" Saccharomyces strains finish the fermentation. [3]

The use of both "ambient" and non-Saccharomyces wild yeasts carries both potential benefits and risk. Some winemakers feel that the use of resident/indigenous yeast helps contribute to the unique expression of terroir in the wine. In wine regions such as Bordeaux, classified and highly regarded estates will often tout the quality of their resident "chateau" strains. To this extent, wineries will often take the leftover pomace and lees from winemaking and return them to the vineyard to be used as compost in order to encourage the sustained presence of favorable strains. But compared to inoculated yeast, these ambient yeasts hold the risk of having a more unpredictable fermentation. Not only could this unpredictability include the presence of off-flavors/aromas and higher volatile acidity but also the potential for a stuck fermentation if the indigenous yeast strains are not vigorous enough to fully convert all the sugars. [3]

It is virtually inevitable that non-Saccharomyces wild yeast will have a role in beginning the fermentation of virtually every wine but for the wineries that choose to allow these yeasts to continue fermenting versus minimizing their influence do so with the intent of enhancing complexity through bio-diversity. While these non-Saccharomyces ferment glucose and fructose into alcohol, they also have the potential to create other intermediates that could influence the aroma and flavor profile of the wine. Some of these intermediates could be positive, such as phenylethanol, which can impart a rose-like aroma. [5] However, as with ambient yeasts, the products of these yeasts can be very unpredictable — especially in terms of the types of flavors and aromas that these yeasts can produce. [3]

When winemakers select a cultured yeast strain, it is largely done because the winemaker wants a predictable fermentation taken to completion by a strain that has a track record of dependability. Among the particular considerations that are often important to winemakers is a yeast's tendency to: [5]

  • Quickly begin fermentation, out-competing other "wild yeasts" for nutrients in the must
  • Completely utilize all fermentable sugars with a predictable sugar-to-alcohol conversion rate
  • Have an alcohol-tolerance up to 15% or even higher depending on the winemaking style
  • Have a high sulfur dioxide tolerance but low production of sulfur compounds such as hydrogen sulfide or dimethyl sulfide
  • Produce a minimum amount of residual pyruvate, acetic acid and acetaldehyde
  • Produce minimum foaming during fermentation which may create difficulties for cap management during maceration or cause bungs to pop out during barrel fermentation.
  • Have high levels of flocculation and lees compaction that makes racking, fining and filtering of the wine easier.

Inoculated (or pure cultured) yeasts are strains of Saccharomyces cerevisiae that have been identified and plated from wineries across the world (including notable producers from well-known wine regions such as Bordeaux, Burgundy, Napa Valley and the Barossa Valley). These strains are tested in laboratories to determine a strain's vigor, sulfur dioxide and alcohol tolerance, production levels of acetic acid and sulfur compounds, ability to re-ferment (positive for sparkling wine but a negative attribute for sweet late-harvest wines), development of surface film on the wine (positive for some Sherry styles but a negative attribute for many other wines), enhancement of a wine's color or certain varietal characteristics by enzymes in the yeast cells and other metabolic products produced by the yeast, foaming and flocculation tendencies, yeasticidal properties (a trait known as "Killer yeast") and tolerance for nutritional deficiencies in a must that may lead to a stuck fermentation. [3]

Re-hydrating freeze dried yeast cultures Edit

Pure culture yeasts that are grown in a lab are often freeze dried and packaged for commercial use. Prior to their addition into must, these yeasts need to be re-hydrated in "starter cultures" that must be carefully monitored (particularly in regards to temperature) to ensure that the yeast cells are not killed off by cold shock. Ideally winemakers want to add enough inoculum to have a viable cell population density of 5 million cells per milliliter. The exact amount of freeze-dried culture varies by manufacturer and strain of yeast but it is often around 1 gram per gallon (or 25 grams per 100 liters). Wines that could have potentially problematic fermentation (such as high sugar level late harvest or botryized wines) may have more yeast added. [5]

Similarly, re-hydration procedures will also vary depending on the manufacturer and winery. Yeast is often inoculated in a volume of water or grape must that is 5–10 times the weight of the dry yeast. This liquid is often brought to temperature of 40 °C (104 °F) prior to the introduction of the yeast (though some yeast strains may need temperatures below 38 °C (100 °F) [1] ) to allow the cells to disperse easily rather than clump and sink to the bottom of the container. The heat activation also allows the cells to quickly reestablish their membrane barrier before soluble cytoplasmic components escape the cell. Re-hydration at lower temperatures can greatly reduce the viability of the yeast with up to 60% cell death if the yeast is re-hydrated at 15 °C (60 °F). The culture is then stirred and aerated to incorporate oxygen into the culture which the yeast uses in the synthesis of needed survival factors. [5]

The temperature of the starter culture is then slowly reduced, often by the graduated addition of must to get within 5–10 °C (9-18 °F) of the must that the culture will be added to. This is done to avoid the sudden cold shock that the yeast cells may experience if the starter culture was added directly to the must itself which can kill up to 60% of the culture. Additionally, surviving cells exposed to cold shock tend to see an increase in hydrogen sulfide production. [5]

In order to successfully complete a fermentation with minimum to no negative attributes being added to the wine, yeast needs to have the full assortment of its nutritional needs met. These include not only an available energy source (carbon in the form of sugars such as glucose) and yeast assimilable nitrogen (ammonia and amino acids or YAN) but also minerals (such as magnesium) and vitamins (such as thiamin and riboflavin) that serve as important growth and survival factors. Among the other nutritional needs of wine yeast: [4]

    – used for the production of nucleic acids, phospholipids (an important component of the cell membrane) and ATP (Adenosine triphosphate which the cell uses for transferring energy for metabolism). – important for the uptake and utilization of phosphate – involved in the synthesis of proteins, fatty acids and nucleic acids. – involved in the metabolism of sugars and lipids. A deficiency of this vitamin could lead into increase hydrogen sulfide production with off-aromas in the resulting wine. – involved in the synthesis of Nicotinamide adenine dinucleotide (NAD+), a co-enzyme that is important in maintaining the redox balance of the cell as well as in the process of ethanol fermentation itself. – involved with the secondary messenger molecules that facilitate cell division.
  • Trace amounts of calcium, chlorine, copper, iron, manganese and zinc for healthy cell function.

Many of these nutrients are available in the must and skins of the grapes themselves but sometimes are supplemented by winemakers with additions such as diammonium phosphate (DAP), freeze-dried micro-nutrients (such as Go-Ferm and Ferm-K) and even the remnant of dead or extracted yeast cells such that the fermenting yeast can break down to mine for available nitrogen and nutrients. One historical winemaking tradition that is still practiced in some Italian wine regions is the ripasso method of adding the leftover pomace from the pressing of other wines into a newly fermenting batch of wine as an additional food source for the yeast. [4]

Saccharomyces cerevisiae can assimilate nitrogen from both inorganic (ammonia and ammonium) and organic forms (amino acids, particularly arginine). As yeast cells die, enzymes within the cells begin autolyzing by breaking down the cell, including the amino acids. This autolysis of the cell provides an available nitrogen source for the still-fermenting and viable yeast cells. However, this autolysis can also release sulfur-link compounds (such as the breakdown of amino acid cysteine) which can combine with other molecules and react with alcohol to create volatile thiols that can contribute to a "stinky fermentation" or later development into various wine faults. [4]

The role of oxygen Edit

Yeasts are facultative anaerobes meaning that they can exist in both the presence and absence of oxygen. While fermentation is traditionally thought of as an anaerobic process done in the absence of oxygen, early exposure of the yeast to oxygen can be a vital component in the successful completion of that fermentation. This is because oxygen is important in the synthesis of cell "survival factors" such as ergosterol and lanosterol. These sterols are important in maintaining the selective permeability of the yeast cell membrane which becomes critical as the yeast becomes exposed to increasing osmotic pressure and levels of alcohol in the wine. As a waste product of its own metabolism, alcohol is actually very toxic to yeast cells. Yeast with weak survival factors and lacking sterols may succumb to these conditions before fermenting a wine to complete dryness, leaving a stuck fermentation. [4]

Cultured yeasts that are freeze-dried and available for inoculation of wine must are deliberately grown in commercial labs in high oxygen/low sugar conditions that favor the development of these survival factors. One of the reasons that some winemakers prefer using inoculated yeast is the predictability of fermentation due to the high level of survival factors that cultured yeast are assured of having without necessarily needing to expose the wine to additional levels of oxygen. Winemakers using "ambient" yeasts that are resident in their winery may not have this same assurance of survival factors and may need to compensate with other winemaking techniques. [4]

Wild non-Saccharomyces yeasts often need a much greater exposure to oxygen in order to build up survival factors which is why many of these yeasts are often found living oxidatively as "film yeast" on the surface of wines in tanks or barrels. [4]

Either directly or indirectly, wine yeast can be a culprit behind a wide variety of wine faults. These can include the presence of "off flavors" and aromas that can be the by-product of some "wild yeast" fermentation such as those by species within the genera of Kloeckera and Candida. Even the common wine yeast Saccharomyces cerevisiae can be behind some wine faults with some strains of the yeast known to produce higher than ideal levels of acetic acid, acetaldehyde and volatile sulfur compounds such as thiols. Also any yeast can have a low tolerance to nutritional deficiencies, temperature fluctuation or extremes and excessive or low sugar levels that may lead to a stuck fermentation. [4]

In the presence of oxygen several species of Candida and Pichia can create a film surface on top of the wine in the tank of barrel. Allowed to go unchecked, these yeasts can rapidly deplete the available free sulfur compounds that keeps a wine protected from oxidation and other microbial attack. The presence of these yeasts is often identified by elevated levels of volatile acidity, particularly acetic acid. Some strains of Pichia will metabolize acetic acid (as well as ethyl acetate and isoamyl acetate that may also be produced) with the side-effect of substantially decreasing the titratable acidity and shifting the pH of wine upwards to levels that make the wine prone to attack by other spoilage microbes. Commonly called "film yeast", these yeasts are distinguished from the flor sherry yeast that are usually welcomed by winemakers in producing the delicate fino-style wines. [4]

Growth of many unfavorable wild yeasts is generally slowed at lower cellar temperatures, so many winemakers who wish to inhibit the activities of these yeasts before the more favorable Saccharomyces yeast kick in, will often chill their must, such as the practice of "cold soaking" the must during a pre-fermentation maceration at temperatures between 4–15 °C (39–50 °F). Though some species, such as Brettanomyces, will not be inhibited and may even thrive during an extended period of cold soaking. [5]

Brettanomyces Edit

The wine yeast Brettanomyces (or "Brett") produces very distinctive aroma compounds, 4-Ethylphenol (4-EP) and 4-Ethylguaiacol (4-EG), that can have a wine being described as smelling like a "barnyard", "wet saddle" or "band-aid". To some winemakers and with some wine styles (such as Pinot noir from Burgundy), a limited amount of these compounds could be considered a positive attribute that adds to the complexity of wine. [4] To other winemakers and with other wine styles (such as Riesling from the Mosel), the presence of any Brett will be considered a fault. [14] Fruit flies are common vector in the transfer of Brettanomyces between tanks and even nearby wineries. [5]

As a fermentation yeast, Brettanomyces can usually ferment a wine up to 10-11% alcohol levels before they die out. Sometimes Brettanomyces already present in a wine that has been inoculated with Saccharomyces cerevisiae will out compete the Saccharomyces strain for nutrients and even inhibit it due to the high levels of acetic acid, decanoic acid and octanoic acid that many strains of Brettanomyces can produce. [5]

Once Brett is in a winery, it is very difficult to control even with strict hygiene and the discarding of barrels and equipment that has previously come into contact with "Bretty" wine. This is because many species of Brettanomyces can use a wide variety of carbon sources in wine and grape must, including ethanol, for metabolism. Additionally, Brett can produce a wide range of by-products that could influence the wine beyond just the 4-EP and 4-EG compounds previously discussed. [4] Many of these compounds, such as the "footprints" of the 4-EP and 4-EG, will still remain in the wine even after yeast cells die and are removed by racking and sterile filtration. [5]


Conclusion

The standardization of wines and beers as a result of the utilization of a genetically reduced set of commercial strains has brought with it the need for new and novel products that can be highlighted and differentiated. In this sense, wild strains and genetically diverse interspecific hybrids are an attractive alternative for the industry. However, challenges persist in adapting and improving wild strains to fermentative environments. Such challenges could be overcome through genetic improvement programs together with adaptive evolution strategies. The generation of new strains and intra‐ and inter-species hybrids could open up new avenues in order to obtain unique strains for the wine and beer industries.


1. Introduction

Saccharomyces cerevisiae (S. cerevisiae) is a unicellular fungus, possessing a nuclear genomic DNA of 12068 kilobases (kb) organized in 16 chromosomes [1]. Its genome has been completely sequenced by Goffeau et al. 1996 [1] and was found to contain approximately 6000 genes, of which, 5570 [2] are predicted to be protein-encoding genes. Bioinformatic analyses have revealed that a number of protein-encoding genes are of foreign origin, i.e., a result of lateral gene transfer, as the term was defined by Doolittle, 1999 [3]. These genes, which entered S. cerevisiae's genome horizontally, are either of prokaryotic or eukaryotic origin [4]. This came initially as a surprise, because of its osmotrophic nutritional style and the presence of robust cell wall, cell- and intracellular membranes. Hall at al., 2005 [4] located 10 genes of putative prokaryotic origin present in S. cerevisiae's genome. One example of acquisition of a gene from another eukaryote is the gene FSY1. FSY1 encodes a fructose transporter [5] and has probably originated from some close relative of S. cerevisiae. This gene is considered as important because its product lends probably to its host strain (EC 1118) an increased capability to utilize fructose under conditions of low hexose concentrations present in the must (i.e., towards the end phase of fermentation).

In respect to S. cerevisiae extra chromosomal elements' genomics, all strains contain of course mitochondrial DNA (mtDNA) molecules, but often with different sizes [6]. The largest version of mtDNA has a length of approximately 85780 bps [7]. Furthermore, most S. cerevisiae strains harbor in their nucleus a distinct extra-chromosomal DNA genetic element called 2µm circle (reviewed by Futcher, 1988 [8]). This double-stranded DNA element has a typical length of 6318 bps and a copy number of approximately 60 copies per cell). It is considered as ‘selfish DNA’ and has nearly no phenotypic consequences for its host, except a slight reduction of the host's growth rate. It is of no use for industrial applications, but on the other hand was highly instrumental for various applications concerning the genetic manipulation of its host. Other extra-chromosomal genetic elements harbored by various strains of S. cerevisiae include single- and double-stranded RNA molecules and retroviruses [9]. Some of these elements have a significant contribution to S. cerevisiae's killer phenotype (s. Section 2.2.2).

S. cerevisiae is a model organism, a valuable tool for all aspects of basic research. Unlike other model organisms though, such as Escherichia coli, or Caenorhabditis elegans, S. cerevisiae is concomitantly also a most valuable species for a variety of industrial applications. One major reason for this feature is one part of its life style, termed ‘make-accumulate-consume’ [10]. This feature is based on the Crabtree effect, which consists in the fact that S. cerevisiae, even under aerobic conditions does not use the respiratory machinery to metabolise saccharides and promote biomass growth, but instead, it produces ethanol and other two-carbon compounds, via pyruvate [11]. The consequence of this fact is that S. cerevisiae produces and accumulates ethanol—which is toxic, or static, for most other microbial species able to compete with it for the sugar compounds- and thus eliminate competition. After S. cerevisiae has cleared the particular ecological niche from most of its competitors, it then proceeds in the consumption of the produced ethanol, thus promoting its own growth. According to Hagman et al., 2013 [12], this strategy evolved gradually before the whole genome duplication of S. cerevisiae and other yeast species, which took place approximately 100 million years ago [13]. It consisted in the loss of a specific cis-acting regulatory sequence (AATTTT) of several promoters, of genes involved in respiration [14]. This sequence is present and conserved in many other yeast genera, such as, Kluyveromyces, Candida and others, while it is absent from the yeast Dekkera, a genus, which includes species, known to be efficient ethanol producers [15]. Certainly, there are two more characteristics, which are very important for some industrial applications of S. cerevisiae: its remarkable resistance/tolerance to high sugar concentrations and production of a number of aromatic, volatile compounds. To the latter characteristic will be devoted special attention during the discussion of vinification.

Environmental strains of S. cerevisiae are subjected to much harsher conditions, than the laboratory ones, which are usually cultured under most favourable conditions. The study of environmental strains reveals, among others, also additional survival strategies developed by this species, which are not apparent during the studies of laboratory/industrial strains. Environmental strains are able to overwinter in the soil, where they can sporulate. Other known natural niches, which S. cerevisiae usually occupies, are leaves and trunks of various plant species, such as oak trees. It is noteworthy, that although S. cerevisiae is found in abundance in environments, such as wineries, its presence there does not originate from grapevines, or grape berries. To the contrary, its presence in the latter habitats is scarce compared to other microorganisms. Mortime and Polsinelli, 1999 [16] determined the frequency of S. cerevisiae's presence in one in a thousand grapes, a frequency much smaller than the ones of other microorganisms. In addition, they found that the incidence of S. cerevisiae increases to one out of four when it concerns damaged grapes in the field. In a different study, Taylor et al., 2014 [17], using a metagenomic approach, were able to detect approximately one S. cerevisiae cell among approximately 20,000 cells belonging to various other fungal genera/species. The rare presence of S. cerevisiae in intact grapes and its much frequenter presence in damaged ones seem to constitute a contradiction, which is explained though by the fact that this organism can occupy an additional niche, i.e., insects. S. cerevisiae is insect-borne, and was detected in several different insects, such as, wasps [18] and Drosophila species [19], which feed on, among others, also on damaged grapes. Stefanini et al., 2012 [18] examined the gut microbiome of social wasps and detect the presence of S. cerevisiae cells, albeit in smaller numbers (4%) compared with other yeasts, such as Candida, or Pichia. Despite its smaller numbers, S. cerevisiae has a stable presence in the wasp community, since it overwinters in the gut of hibernating colony founding queens from autumn until spring and then, is transferred to their larvae through feeding. Regarding the Drosophila - S. cerevisiae interaction, Buser et al., 2014 [19] in the course of their study of the niche construction theory, showed that Drosophila simulans has a preference for yeast producing more efficient attractants. This is a mutually beneficial interaction, because while the flies that harbor the yeast exhibit an increased fecundity, S. cerevisiae benefits from being transferred to new niches, such as damaged grapes. This explains the higher incidence of S. cerevisiae in damaged grapes, compared with the incidence on intact ones. The frequency of S. cerevisiae occurrence in the environment is still under study, but it is much frequenter than initially anticipated. Wang et al., 2012 [20] collected 2064 samples from various natural, not human-made, habitats in China and were able, using an enrichment-based approach, to detect the presence of S. cerevisiae in 226 of them (10.9 %). The genetic diversity of S. cerevisiae isolates found in the positive samples was also much larger than in human-made, or human–‘influenced’ ones. This could be potentially very important, because environmental, ‘wild’, strains could bear genotypes with highly interesting properties for biotechnological applications. The use of ‘wild’ strains for industrial applications though may not be a simple procedure, because the genetic diversity does not always correspond to a phenotypic one. Camarasa et al., 2011 [21] e.g., studied the efficiency of 72 S. cerevisiae strains of diverse origins (industrial, laboratory, environmental) under conditions of must fermentation and found that strains originating from rich in sugar environments were able to finish the fermentation process, while the laboratory or environmental strains were unable to perform satisfactorily. The molecular basis of the better adaptation, especially of the wine strains, to the stressful conditions of must fermentation is yet unknown and could be attributed more than one reasons such as epigenetic phenomena.

This review focuses mainly on various aspects of industrial applications employing S. cerevisiae, especially those related to its fermentation capacity and its use in the wine and food industry, as well as in the bioethanol production. Furthermore, we present data highlighting the potential of environmental S. cerevisiae isolates in the above-mentioned biotechnological applications.


Why, When, and How to Measure YAN

By managing fermentation, winemakers today have many options to enhance the varietal characteristics of their wines, and to express regional attributes. Winemakers know that temperature is a management tool that affects the rate of fermentation similarly, the presence of grape solids enhance yeast survival. Very importantly, adequate nitrogen (N) is necessary for a successful fermentation.

Grapes contain a variety of nitrogenous compounds, the sum of which may be affected by viticultural practices. For instance, research has demonstrated that N concentration is 2X greater with application of foliar N and appropriate irrigation use than without foliar N and irrigation. Other research suggests that N application around veraison appears to be an effective way increasing N in the fruit, regardless of water-supply status of the vines. The concentration of nutrients, whether too great or too little, can induce stress and lead to different concentrations of flavor compounds. For instance, H2S formation is a well-known example related to inadequate nutrients leading to nitrogen depletion stress.

A common practice among winemakers is to make a standard addition of diammonium phosphate (DAP), or other N source, to the juice or must (100-300 mg/L) at inoculation without measuring the nitrogen concentration. The objective of this article is to show that N addition has significant flavor (and ultimately, economic) consequences and that measuring the initial nitrogen concentration provides the opportunity to adjust N addition – not only to achieve an adequate fermentation rate, but also to more reliably guide the flavor profile and style of wine intended.

Definition and Measurement of “YAN”

Grapes contain a variety of nitrogenous compounds of which the most important are the primary (alpha) amino acids, ammonium ions, and small peptides. These three nitrogenous compounds – amino acids (excluding proline), ammonium ions, and small peptides – constitute what is commonly referred to as yeast assimilable nitrogen (YAN) or fermentable nitrogen.

YAN measurements, ideally, should be performed directly on juice or must samples at the point of inoculation to avoid over-estimation due to processing losses which inevitably occur between vineyard and the fermenter. Juice samples taken from grape musts can under-estimate total berry YAN due to the disproportionate concentration of amino acids contained in the unsampled grape skins. While an early warning for low YAN may be obtained by sampling in the vineyard one to two weeks prior to harvest, measurement immediately before fermentation is necessary due to the highly variable nature of YAN measurements during those last weeks before harvest.

Favored methods of measurement of YAN are (a) enzymatic assay kits, (b) the method known as the Formol Titration, which consists of neutralizing a juice sample with a base, then adding an excess of neutralized formaldehyde, and re-titrating the resulting solution to an endpoint and (c) use of expensive equipment such as the HPLC (high-performance liquid chromatography). Typically, wineries use the first two methods commercial labs may use the third method.

How much YAN

YAN has the most impact on fermentation speed compared to other compounds. It impacts yeast biomass at the beginning of fermentation and sugar transport during fermentation. At the end of growth phase, N is depleted resulting in decreased protein synthesis and sugar transport. A YAN addition at this point reactivates protein synthesis and sugar transport increasing the fermentation rate. Oxygen is rapidly consumed in the beginning of fermentation. Decreased oxygen inhibits sterols and fatty acid synthesis by yeast. This causes decreased yeast growth and viability at the end of fermentation.

Sterols and fatty acids are survival factors needed for the yeast cell membrane to function. As ethanol increases, hydrogen ions accumulate in cell requiring more energy to expel them. The pH decreases inside the cell causing cell death. Oxygen adds at end of growth phase increase sterol production. Therefore, microoxygenation and pump overs would be beneficial at 1/3 of the way through alcoholic fermentation (end of yeast growth phase).

Budding Saccharomyces cerevisiae photo by Molly Kelly

N assimilation

The manner in which N is assimilated by yeast depends on the source. Organic N (amino acids) is actively transported into the yeast cell. Through additional reactions N is incorporated into glutamine and glutamate and eventually used in the synthesis of other amino acids and nitrogenous compounds. This process is gradual and efficient compared to inorganic sources. Ammonium nitrogen (inorganic N) is consumed quickly and is less beneficial. Amino acid mixtures vs single N sources are more efficient because the yeast directly incorporates the amino acids into proteins rather than having to synthesize them.

Ammonia, which exists as ammonium (NH 4 +) ions in must, is used by yeasts prior to amino acids. The presence of NH 4 + delays timing and uptake of amino acids by yeast.

The timing of N supplements and form of supplement will impact fermentation and volatiles. Types of N supplements include Diammonium phosphate (DAP), proprietary blends of DAP and amino acids (e.g. Superfood®, Fermaid K®, Actiferm) and balanced nutritional formulas containing inorganic N (e.g. Fermaid O®), organic N, sterols, yeast cell walls, fatty acids, yeast autolysis products and others. DAP is best used with low N musts. Other balanced nutrients should be added as well. At a rate of 100 mg/L DAP, 20 mg/L YAN is added.

It is common practice for winemakers to make N additions at the following times:

  • Yeast rehydration to rebuild cell walls (rehydration nutrients consist of inactivated yeast and autolysates. They contain no inorganic N and only 3 mg/L N for every 100 mg/L added).
  • Six-twelve hours after inoculation (2-3 Brix drop)
  • End of growth/exponential phase (1/3 sugar depletion)

Yeast Growth throughout Fermentation

Note that at ½ sugar depletion the yeast cannot utilize N since alcohol accumulation prevents uptake. This residual N can then be utilized by other organisms such as Brettanomyces spp.

Results of Deficient YAN

From a practical point of view, the problem of juice nitrogen composition is primarily linked to juices with suboptimal concentrations of nitrogen (<150 mg/L), and higher risk of slow or stuck fermentation. Low YAN (< 200 mg/L) is associated with production of sulfur compounds, e.g. hydrogen sulfide, which results from the nitrogen demand for yeast growth. The amount of H2S produced is dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the enzymatic activity immediately before nitrogen depletion.

When working with very low YAN juices, researchers have observed that other nutrients can also be low. Therefore, when YAN is low and other nutrient deficiencies are suspected, it may be useful to add a proprietary yeast food that contains more complex forms of N, as well as vitamins, lipids and minerals. Continued H2S production after N addition suggests a general vitamin deficiency, though other causes are also possible. Most yeast suppliers can advise on the use of yeast foods, which are generally produced from inactivated yeast, e.g. GoFerm® or similar additives.

In summary, low must YAN leads to low yeast populations and poor fermentation vigor, increased risk of sluggish/stuck/slow fermentations, increased production of undesirable thiols (e.g. hydrogen sulfide) and low production of favorable sensory compounds including esters and long chain volatile fatty acids.

Results of Excessive YAN

High must YAN leads to increased biomass and higher maximum heat output due to greater fermentation vigor. Overuse of DAP can also stimulate overproduction of acetate esters, especially ethyl acetate, resulting in the perception of volatile acidity (VA) and suppression of varietal character. High YAN (exceeding 450-500mg/L YAN) can stimulate ethyl acetate production by many yeast strains. Increased concentrations of haze-causing proteins, urea and ethyl carbamate and biogenic amines are also associated with high YAN musts. The risk of microbial instability, potential taint from Botrytis-infected fruit and possibly atypical aging character is also increased.

Main Flavor Changes Affected by Nitrogen

In general, YAN can affect TA and the balance of organic acids which can affect flavor. Malic acid consumption increases with increasing DAP concentration, irrespective of yeast strain. When total nitrogen is increased by adding ammonium to a medium containing very low levels of YAN, the production of higher alcohols is initially increased, but then tends to decrease after a peak between 200-300mg/L YAN. This activity depends on various factors, including yeast strain and fermentation conditions. Higher alcohols are characterized by fusel-like odors, and are generally thought to contribute to the complexity of wine fermentation bouquet. However, when present in very high concentrations they can have a negative impact on wine aroma, mainly because they mask fruity characters.

Of course, intermediate must YAN favors the best balance between desirable and undesirable chemical and sensory wine attributes. The key is to have timely and accurate YAN must concentration data immediately before primary inoculation. Recognizing that measurement is difficult in a winery setting, we encourage use of commercial and extension labs that offer YAN measurements, so that the winemaker might make an informed decision regarding supplemental nitrogen additions.

AWRI: Maurizio Ugliano, P. A. H., Markus J. Herderich, Isak S. Pretorius. 2007. Nitrogen management is critical for wine flavour and style. AWRI Report: The Australian Wine Research Institute, vol. 22. Wine Industry Journal, Glen Osmond (Adelaide), South Australia 5064, Australia.

Barthe, C., M. Dorais, G. Dubé, P. Angers, and K. Pedneault. 2013. Abstracts from Presentations at the ASEV–Eastern Section, 2013, Winston-Salem, NC. 64:417A.

Bell, S.-J., and P. A. Henschke. 2005. Implications of nitrogen nutrition for grapes, fermentation and wine. Australian Journal of Grape and Wine Research 11:242-295.

Blateyron, L. O.-J., A Sablayrolles, J.M. 2003. Stuck fermentations: oxygen and nitrogen requirements – importance of optimising their addition. Aust. N.Z. Grapegrower Winemaker:73-79.

Cheng, L., T. Henick-Kling, A. Lakso, and T. Martinson. 2003. Abstracts, ASEV Eastern Section 27th Annual Meeting, 2002, Baltimore, MD. American Journal of Enology and Viticulture 54.

Henschke, P. A. 1996. Presented at the Eleventh international oenological symposium, Sopron, Hungary.

Henschke, P. A. J., V. 1993. Yeasts – metabolism of nitrogen compounds, p. 77-164. In G. H. Fleet (ed.), Wine Microbiology and Biotechnology. Harwood Academic Publishers, Chur, Switzerland.

Leonardelli, Michael J. Enology News & Notes, Volume 3, #2, ICCVE, U of Missouri, Fall/Winter 2013-2014

Moundtop.com. 2011. Estimation of Yeast Assimilable Nitrogen using the Formol Titration Technique, p. 3. Version 1.1 ed. http://www.moundtop.com.

Ribereau-Gayon, J. D., D. Doneche, B. Lonvaud, A. 2000. Handbook of Enology, Volume 1: The microbiology of wines and vinification, vol. John Wiley & Sons Ltd:, Chichester, UK.

Kelly, M., G. Giese and B. Zoecklein. Abstracts, Poster Session, Nitrogen Symposium, ASEV 66th National Conference, 2015, Portland, OR.


Brewing With Wine Yeast

Although Saccharomyces cerevisiae was so named for beer because it was first identified at a brewery, yeast strains from the species have been fermenting juice into wine for nearly as long. Brewing inevitably became more industrialized and scientific than winemaking because beer ingredients could be transported and stored for centralized production before refrigeration. Even with the explosion of craft breweries, today there are still 2,000 more wineries in America, yet these wineries produce only 1⁄7 the volume (27,514,101 bbls of wine 1 compared to 189,839,914 bbls of beer 2 in 2016). If winemaking had been the more industrialized beverage we might be pitching Saccharomyces vinum into our wort.

The (often inaccurate) winemaker stereotype is a refined person in a crisp shirt strolling through their rustic vineyard in contrast to the tattooed brewer in a grungy urban factory. However, when it comes to the yeast in their employ, it is wine yeast that is less genteel. Most strains of beer yeast have been living in breweries year-round for centuries, leaving the yeast pampered without need to compete with wild strains. On the other hand, commercial wine strains continued to overwinter in the vineyards until relatively recently, causing many wine strains to retain their ability to kill competing yeast.

Wine strains are selected for different reasons and adapted to different conditions than beer yeast. Their resulting unique characteristics can be beneficial for brewing in the right context. The most common use of wine yeast in beer is for bottle conditioning, where their high alcohol and acid tolerance allow it to easily carbonate a barleywine or Flemish red. Wine yeasts were selected to enhance the fruit flavors of the grapes and so can free, alter, or reinforce the same compounds contributed by hops or fruit. However, wine yeasts’ subsistence on the glucose and fructose in juice result in most ill-equipped to ferment the complex starch-derived carbohydrates in wort. While there is potential for beer fermented by wine yeast, there are many considerations before pitching them into wort.

General Considerations

Similar to brewer’s strains, wine yeasts ferment sugar into ethanol and carbon dioxide. However, their long-term diet of simple sugars means that most wine strains didn’t develop as many copies of the genes responsible for transporting and splitting maltose and maltotriose. 3 As a result, almost all wine strains struggle to surpass 50% apparent attenuation in a standard wort. One reported exception is Lalvin ICV K1-V1116. Low attenuation is an obvious hindrance but also can be turned into an advantage in certain situations, like sour beers. It is clear that wine strains are usually not ideal choices to pitch into an under-attenuated imperial stout!

There are a few options to mitigate the attenuation issue. The first is to produce a highly-fermentable wort. This approach could include an extended mash held at a temperature favoring beta-amylase (145 °F/63 °C) potentially augmented with simple sugars in place of 10-20% of the malt. The same goal can be accomplished with the addition of commercial enzymes amylase powder or Beano® can even be added to the fermenter.

Another approach is to use a standard wort and pitch a blend of wine and brewer’s yeast, or stagger them allowing the beer strain to finish what the wine yeast started. This process isn’t as simple as it is for teaming-up two brewer’s yeasts, as I wrote about a few years ago (“Collaborative Fermentations” – BYO March/April 2015). With wine yeast there is an additional factor to consider. Many wine yeasts produce a “kill factor” protein capable of incapacitating susceptible yeast, including almost all beer strains. There are different versions (e.g., K1, K28), and while they attack cells in a variety of ways the end result is much the same. Rather than being considered a detriment, many winemakers prefer killer strains because they defend the wine from other unwanted wild Saccharomyces present in the must.

Most yeast labs list whether or not a strain is a killer or not, 4 so consider a sensitive or neutral strain (e.g., Lalvin 71B) for co-fermentation with brewer’s yeast. If that isn’t an option, pitch less than 2.5% killer yeast and the sensitive strains will be unaffected. 5 The same study also notes that the peak activity of the kill factors is between 4.2 and 4.7 pH, which is the usual final pH of beer.

Wine strains benefit from the same sorts of conditions in terms of aeration, nutrients, and pitching rate as beer yeast. Their ideal temperature has a similar range to brewer’s yeast with white wine strains thriving as cool as lager yeast (as low as 50 °F/10 °C) and red wine strains as warm as saison yeast (as high as 95 °F/35 °C). In Farmhouse Ales (Brewer’s Publications, 2004), Phil Markowski even theorizes that the Saison Dupont yeast may have mutated from a red wine strain given the similar temperature requirements. Consult the yeast lab’s recommended temperature range for the strain as a starting point.

Between 81-95% of wine yeasts produce phenolic aromatics, 6 similar to Belgian, hefeweizen, and many wild Saccharomyces strains. These can range from clove to smoky to rubbery. Wort contains more of the precursors including ferulic acid (1-7 mg/L 7 ) than grapes (<1 mg/L 8 ), especially when wheat malt is added or a low-temperature ferulic acid rest is used. The lack of ferulic acid in grape juice is one reason that we usually don’t taste high-levels of pepper or clove in wine.

Interactions with Hops

Many sophisticated drinkers compare the character of Nelson Sauvin hops to New Zealand Sauvignon Blanc wine, but this isn’t just beer-judge banter both contain 3-sulfanyl-4-methylpentan-1-ol (3S4MP) and 3-sulfanyl-4-methylpentyl acetate (3S4MPA) which provide some of their shared citrusy aromatics. 9 3S4MP is also found in high concentration in Mosaic® and Hallertau Blanc hop varieties. 10 There are compounds that other hops and grapes have in common as well. In addition, some “varietal enhancing” wine yeasts are fantastic for freeing bound hop aromatics from water-soluble glycosides (“The Science of Hop Glycosides” – BYO July/August 2015). As a result, wine yeast may have a modifying or enhancing effect on hoppy beers!

Much of the interaction happens during the yeast’s growth phase, so the hops added to the boil are essential. You might choose specific varieties and additions to accentuate a particular contribution. For example, I hopped an IPA with Simcoe® late in the boil because the concentration of the thiol 3-mercaptohexan-1-ol (3MH), reminiscent of grapefruit and passion fruit, increases with up to 20 minutes at a boil (while catty 4MMP decreases). 11 Certain wine yeasts have the ability to convert 3MH to 3MHA (similar tropical and citrus flavors but with a sensory threshold at 4 ng/L compared to 60). 12 In addition to a standard pitch of brewer’s yeast for 5 gallons (19 L), I pitched 1 g of Anchor Alchemy II, a yeast blend developed by Anchor Wine Yeast with that specific conversion in mind for South African wines. The passion fruit aromatics didn’t jump out compared to the half of the batch without wine yeast, but the wine yeast did create a brighter more saturated citrusy hop flavor.

Sour Beer Primary Fermentation

One consideration for mixed-fermentation sour beers is leaving enough carbohydrates (often in the form of dextrins) for the bacteria (especially Pediococcus) to create lactic acid. Often this result is accomplished with a hot mash temperature, creating wort that is unsuitable for a clean brewer’s yeast fermentation. Wine yeast, being less attenuative, can be used for primary fermentation before or in conjunction with Brettanomyces, Lactobacillus, and Pediococcus. Luckily, none of these are sensitive to kill factors and the Brett will change the phenols created. I have found sour beers primary fermented with wine yeast (including Lalvin BM45) to retain some of their interesting aromatics. Consider a strain that produces high-levels of glycerol (e.g., Lalvin S6U) to enhance mouthfeel.

Like hops, fruits other than grapes can provide compounds for wine yeast interaction. The enzymes used by the yeast have an optimum pH higher than a typical finished sour beer though. Usually I wait to add fruit until a couple months before bottling to give my trial the best chance to succeed I had to change tacks. I brewed a Flemish red with dried sour cherries (1 lb. in 5 gallons/0.45 kg in 19 L) added after a Danstar 58W3 (Viti Levure) primary fermentation. The dried fruit provided a pervasive cherry-backdrop, enhanced by the wine yeast. After souring on the dried fruit for six months I transferred the beer onto homegrown sour cherries and local dark cherries for a final burst of fresh fruit aroma (Recipe found here). The combination was magical, one of the best sour beers I’ve brewed!

Start Wine-ing

There are a multitude of wine strains available, but not a lot of documented experience when it comes to fermenting beer with them or analysis of their interactions with hops and non-grape fruit. Like most experiments, the easiest way to begin is to divert a small percentage of wort to reduce the risk and allow for comparison. See what strains work with which ingredients, and share your experiences with the rest of us! Wine yeast are often inexpensive, so you are only risking a couple dollars with the payoff being unique flavors, better mouthfeel, and a signature character that may be surprisingly wonderful!