Difference between revisions of "Brettanomyces"

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Brettanomyces

Brett Aroma Wheel by Dr. Linda Bisson and Lucy Joseph at UC Davis

Brettanomyces, also referred to by brewers as "Brett" or "Bretta", is Greek for "British Fungus" and is a yeast that was originally thought of as an important yeast for producing the character of some 17th century and prior English ales. Since the wide adoption of pure cultures of Saccharomyces cerevisiae and S. pastorianus in the brewing and wine industries starting in the late 1800's, Brettanomyces has been mostly viewed as a spoilage yeast, except in Belgian lambic, Flanders red/brown beers, and a handful of styles of wine. More recently Brettanomyces has gained popularity in the United States (and subsequently the brewing industries of other countries) as a yeast that can contribute desirable and novel characteristics to beer and other alcoholic beverages. The genus name Dekkera is used interchangeably with Brettanomyces, as it describes the teleomorph or spore forming form of the yeast ^[1]. Known for it's barnyard, fecal, horsey, metallic or Band-Aid flavors, Brettanomyces continues to be unwelcome in many breweries and most wineries. However, Brettanomyces also produces high levels of fruity esters that are desirable in some styles like saison, lambic, and American sour beers. Brettanomyces can also form a pellicle during fermentation. See Lactobacillus, Pediococcus, Saccharomyces, and Mixed Cultures charts for other commercially available cultures.

Introduction of Characteristics and Taxonomy

Although first isolated in 1889 and again in 1899 by scientists at Guinness, the discovery of Brettanomyces was first publicly published by Hjelte Claussen in 1904 after he cultured it in 1903 from English beers that exhibited a sluggish secondary fermentation ^[2]. At the time of discovery, Claussen was aiming to recreate the flavor profile of traditional English ales by fermenting them with pure cultures of Saccharomyces, and either pitching pure cultures of his newly discovered Brettanomyces yeast along with Saccharomyces, or as he preferred, after the primary fermentation of Saccharomyces ^[3]. Although Claussen saw the character from Brettanomyces as a desirable character in ales and identified its character as a quality of traditional English ales, at some point Brettanomyces became identified as a contaminate in wineries and breweries due to some of the phenols, acids, and haze that it sometimes produces. These phenols and acids have generally been described as "barnyard", "burnt plastic", "wet animal", "fecal", and "horse sweat", although some tasters describe these flavors with different terminology because they perceive certain flavor compounds differently while some other tasters simply cannot detect certain flavor compounds at all ^[4]^[5]^[6]. The general viewpoint of brewers (other than Lambic and Flanders red/brown brewers in Belgium) and vintners that Brettanomyces is primarily a spoilage organism held for many decades, and still holds in most cases. More recently the positive flavor components that have been identified in Brettanomyces beer such as pineapple, stone fruits, and to some degree acetic acid, have regained popularity with brewers outside of Belgium. Wine tasters have also described wines with certain flavor compounds derived from Brettanomyces as positive characteristics of wine. It is important to keep in mind that individual tasters on tasting panels describe some flavor compounds as "negative" while others describe them as "positive" (and sometimes a mixed response is given by a taster in regards to a certain flavor compound), and this discrepancy in interpretations of Brettanomyces derived flavor compounds appears to be based on personal preference and experience. In some cases, for example for vinyl phenols, low levels that are not detectable by some people, but detected by others contribute positively to wine, while higher amounts contribute negatively. Thus, lower intensity of some flavor compounds can be seen as more desirable. Overall, the enjoyment or displeasure of the various flavor compounds produced by Brettanomyces and at certain levels is completely subjective ^[6].

It is common in scientific literature to see the names Dekkera and Brettanomyces used as the genus name, with Dekkera being the teleomorph version and Brettanomyces being the anamorph. There are five species within the genus of Brettanomyces: B. anomalus, B. bruxellensis, B. custersianus, B. nanus, and B. naardenensis (one study on the genetics of B. nanus from 1990 classified B. nanus as belonging to another genus of yeast called Eeniella, however this has not been agreed upon in more recent studies ^[7]). The species known as B. intermedius and B. lambicus are considered synonyms of B. bruxellensis ^[8]. Of these five species, only B. anomalus and B. bruxellensis have been identified to have a teleomorph version. In their teleomorph version they are referred to as Dekkera anomala and Dekkera bruxellensis ^[4]^[5]^[9]^[10]. All of the other names such as the ones often used by yeast labs are derived by old nomenclature that is no longer used (click here for a table that lists old and new taxonomical nomenclature). Most Brettanomyces cultures from brewer's yeast labs are classified genetically as B. bruxellensis or B. anomalus.

The morphology of Brettanomyces can vary immensely from strain to strain (and species to species). Some strains can look similar in size and shape to S. cerevisiae under a microscopic image, while others are elongated or much smaller. This makes it difficult to identify Brettanomyces without DNA analysis. Morphologies of Brettanomyces grown on agar plates can also be different from strain to strain. For example, Devin Henry found that a sample of WLP648 that contained two closely related strains of B. bruxellensis grew completely differently on the same growth media. At first, larger, slightly off-white colonies grew on the plates (this was the first strain), and then a few days later the second strain grew as many smaller white-colored colonies. Other strains may appear as glossy or matted with jagged edges, etc. Morphology on agar plates can change depending on the type of growth media ^[11]^[12]^[13].

Recently a new species of Brettanomyces has been proposed, although classification has not been fully established. The proposed name is Brettanomyces acidodurans sp. nov. Two strains of B. acidodurans were isolated from olive oil from Span and Israel, however, its presence in olive oil has been described as "rare" because only two strains were found after searching dozens of olive oils. Its closest relation is to B. naardenesis by 73% of its genetic makeup. No teleomorph form was observed. This species is a strong acetic acid producer, and it is very tolerant of acetic acid in its environment. It can consume lactose and cellobiose but does not consume maltose. it is unknown but a possibility that this species contributes to the vinegary taste of spoiled olive oils, although this has generally been attributed to acetic acid bacteria ^[14].

Environment and Survival

Brettanomyces has been occasionally identified on the skins of fruit (on the skins of cider apples and wine grapes for example) ^[4]^[5], and is thought to disperse via insects (called "vectors" in the scientific literature) ^[9]. However, the occurrence of Brettanomyces has more commonly been identified in industrial food processing areas (wine, beer, kombucha, soft drinks, dairy products, tea, sourdough, etc.) ^[15]. Brettanomyces is not considered to be airborne, however studies have found a very small amount of cells in the air at wineries where wine with Brettanomyces in it was being handled (most of the yeasts found in the air were Aureobasidium and Cryptococcus, which aren't considered spoilage organisms in beer and wine). These set of studies also determined that very specific methodology was needed in order capture Brettanomyces from the air, and indicated that the yeast was "stressed". ^[16]. While it is possible for Brettanomyces to be briefly carried by gusts of air, it only happens in the vicinity where the Brettanomyces beer or wine is being bottled (more so) or is actively fermenting (less so). Good cleaning and sanitation and cold temperatures should be employed to keep Brettanomyces from contaminating other equipment, however flying insects are also a likely cause for contamination of Brettanomyces.

Brettanomyces is commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries. These include structures such as wooden fermentation vessels, walls of the building, as well as the inside surface of wood barrels and actually buried within the wood of barrels up to 8 mm ^[8], with the highest concentration of surviving cells being at the top staves where oxygen is more accessible. Some strains are able to utilize the cellulose of the wood as a carbon source. Ozone has been shown to be an effective way to kill Brettanomyces that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing Brettanomyces within the wood of barrels ^[17]. Although the role of Brettanomyces appears to be limited in distillation, it has been isolated during the fermentation process of tequila making. It has also been isolated from drains, pumps, transfer hoses, and other equipment that is difficult to sanitize. The survivability of Brettanomyces has also partly been attributed to its ability to form a biofilm (in particular B. bruxellensis). Microorganisms that can form a biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. Brettanomyces has therefore been identified as a significant contaminate for breweries and wineries. Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for B. bruxellensis. Brettanomyces is also commonly found in sherry, and is found (although only rarely) in olive production, lemonade, kombucha, yogurt, pickles, and soft drinks. B. anomalus and B. bruxellensis are generally found much more commonly than the other three species of Brettanomyces. ^[4].

Unlike most genres of yeast, Brettanomyces has the characteristics of being very tolerant to harsh conditions, including high amounts of alcohol (up to 14.5-15% ABV ^[18]^[8]), a pH as low as 2 ^[19], and environments with low nitrogen ^[5]. This capability allows Brettanomyces to survive in alcoholic beverages such as beer, wine, and cider. In alcoholic beverages, B. bruxellensis tends to lag after the primary fermentation with Saccharomyces. It is believed that during this lag phase, B. bruxellensis adapts to the harsh conditions of the beverage (low pH, high concentrations of ethanol, and limited sugar/nitrogen sources). After this lag phase, B. bruxellensis can grow and survive when no other yeasts can. Brettanomyces is also more resistant to pH and temperature changes, and tolerant of environments limited in oxygen (although Brettanomyces prefers the availability of at least a little bit of oxygen). Scientifically, which specific nitrogen and carbon sources B. bruxellensis uses in these stressful environments has not received much research ^[4].

The genetic diversity of Brettanomyces is particularly wide. Some studies have indicated that strains of B. bruxellensis have adapted to specific environments. For example, one study found that strains of B. bruxellensis isolated from wine had 20 genes involved in the metabolism of carbon and nitrogen, whereas strains isolated from beer did not. This indicated that B. bruxellensis strains living in wine have adapted to the harsher environment of wine ^[4]. Another study found that one out of the two strains tested that were isolated from soda could not ferment maltose, and only strains isolated from wine were able to grow in wine and the beer/soda strains did not. The wine strains were also more resistant to sulfites, which are commonly used in the wine industry to prevent microbial contamination ^[15].

Sulfite and SO₂ inhibits the growth of Brettanomyces, and is often used in the wine industry to prevent the growth of Brettanomyces (some wineries have identified small amounts of flavors from Brettanomyces as being beneficial to certain wine styles, and is said to increase the complexity and impart an aged character in young wines ^[4]) ^[20]. However, it has been shown that wine strains of B. bruxellensis could survive dosages of up to 1 mg/L of SO₂, and the very high dosage of 2.1 mg/L was needed to kill Brettanomyces in wine ^[8]. This dosage of SO₂ is beyond legal limits and has negative effects on wine. The tolerance of SO₂ of strains of Brettanomyces found in beer is lower than those found in wine strains. Some strains of Candida pyralidae, Wickerhamomyces anomalus, Kluyveromyces wickeramii, Torulaspora delbrueckii and Pichia membranifaciens have been found to produce toxin that inhibits Brettanomyces, and these toxins have been proposed as an alternative to SO₂ as a way to kill Brettanomyces (killer wine strains of Saccharomyces cerevisiae do not kill Brettanomyces; see Killer Wine Yeast for more information).

The addition of vitamins can have a positive impact on Brettanomyces growth. For example, while Brettanomyces does not need riboflavin (vitamin B2) or thiamine (vitamin B1) in order to grow, the presence of either or both of these two vitamins encourages Brettanomyces growth ^[21].

Brettanomyces Metabolism

Like Saccharomyces, Brettanomyces is Crabtree positive (produces alcohol in the presence of oxygen and high sugar concentration), and is petite positive (unable to grow without carbon sources, and forms small colonies when able to grow on growth media) ^[4]. Perhaps the most differentiating characteristic of Brettanomyces is its preference to ferment glucose in the presence of oxygen to produce ethanol and acetic acid, which is the opposite preference in Saccharomyces where the presence of oxygen inhibits fermentation (dubbed the "Pasteur effect"). Also opposite of most yeasts including Saccharomyces, in a completely anaerobic environment Brettanomyces ceases alcoholic fermentation for about 7-8 hours before adapting to the anaerobic conditions ^[8]. This was initially dubbed the "negative Pasteur effect" by Custers, and later the "Custers effect" by W. A. Scheffers ^[22]^[23]. Despite the Custers effect in Brettanomyces, this genus is not classified as an "oxidative yeast" but rather as a "fermentative yeast" since oxidative yeasts produce little to no ethanol in the presence of glucose, and only grow on the surface of a liquid rather than within a liquid ^[24]^[25]^[26]^[27]^[28].

Carbohydrate Metabolism and Fermentation Temperature

Brettanomyces is able to ferment a wide range of sugars. All strains can ferment glucose, and many strains can ferment sucrose, fructose, and maltose, although at a slower rate than glucose. Some strains can also ferment galactose, mannose, ethanol, acetic acid, and glycerol, although there are some contradicting studies in science regarding the specifics, and many previously published studies do not specify whether testing conditions were aerobic or anaerobic even though the availability of oxygen effects whether or not certain sugars can be fermented by a given strain of Brettanomyces ^[9]^[4]. Acetic acid, glycerol, succinic acid, and ethanol are only consumed if oxygen is present ^[4]. The addition of small amounts of O2 stimulates glucose fermentation, as well as H+ acceptors such as acetaldehyde, acetone, pyruvic acid and other carbonyl compounds ^[22].

Brettanomyces strains may possess both alpha and beta-glucosidases. These enzymes allow Brettanomyces strains to break down a broad range of sugars, including long-chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to Saccharomyces yeasts. ^[9]^[29].

Extracellular and intracellular alpha-glucosidase activity has been shown to break down sugars up to 9-12 chain carbons in one strain of B. lambicus (now classified as B. bruxellensis), which is partly responsible for the slow, over-attenuation of wort that some strains of Brettanomyces an achieve in beers such as lambic and American sour beers ^[22]^[4]. Alpha-glucosidases are the enzymes that allow them to break down maltose, turanose, melezitose, and trehalose, as well as dextrins such as maltotetraose and maltopentaose. These enzymes work by cleaving off glucose that can be directly consumed by the cell, leaving a shorter chain sugar behind which is then further broken down. In the case of extracellular alpha-glucosidase activity, this breakdown of complex sugars occurs outside of the cell and may benefit other microorganisms if present such as lactic acid bacteria. These dextrins are left over after a normal Saccharomyces fermentation ^[9]. Some other polysaccharides can be fermented by Brettanomyces, including starch, laminarin, and pectin ^[18]. The more complex the starch or sugar, the slower it is hydrolyzed by the alpha-glucosidase enzymes. The optimal pH for the alpha-glucosidase enzyme produced by one strain of B. bruxellensis was 6 and at a temperature of 39-40°C (102-104°F), and its activity was greatly reduced below a pH of 4.5 and above 8 (although citric acid was used as a buffer, and its effects on the enzyme was not compared to other acids), which might contribute to slower Brettanomyces fermentation in acidic beers ^[30].

Beta-glucosidases can break down the beta-glycosidic bond in disaccharides (cellulose, cellobiose, and gentiobiose) ^[31]^[4], as well as glycosides. Glycosides are sugar molecules connected to other organic compounds such as acids, alcohols, and aldehydes which are flavor and aroma inactive due to the sugar molecule attached. By cleaving off the sugar molecule through beta-glucosidase activity, Brettanomyces species can liberate these compounds (called aglycones) into their aroma-active and flavor-active states, or states that may become flavor and aroma active through further modification ^[32]. Therefore some Brettanomyces strains are believed to be able to produce novel flavors and aromas from hops, fruits, and fruit pits that Saccharomyces yeasts cannot produce. In addition, the liberated aroma and flavor active compounds may be further processed by Brettanomyces through ester production or destruction pathways. See Beta-Glucosidase Activity for more information.

There is a highly genetic diversity between strains of Brettanomyces species, both in a genotypic and phenotypic sense ^[18]. Not all species are capable of consuming the same types of sugars. For example, B. anomalus (aka claussenii) are generally able to ferment lactose, but B. bruxellensis is generally not. Different strains within the same species may not be able to ferment the same types of sugars ^[33]^[34]. For example, some strains are not able to ferment maltose (often B. anomalus strains), which is almost half the sugar content of wort ^[35]. Such strains would not be a good choice for 100% Brettanomyces fermentation.

The ability of a given Brettanomyces strain to ferment different types of sugars might be at least partially linked to its source of isolation. For example, a strain of B. bruxellensis isolated from a soft drink could not ferment the disaccharides maltose, turanose, or the trisaccharide melezitose, whereas all of the other B. bruxellensis strains isolated from beer and wine could ferment these disaccharides/trisaccharide. The beer strains, however, were unable to ferment cellobiose or gentiobiose, as well as arbutin and methyl-glucoside. The wine strains were able to ferment these disaccharides, perhaps because they were adapted to the environment in which they were isolated from (wine barrels). Further studies are needed to see if this is a trend throughout the species ^[18]. A study by Tyrawa et al. from Escarpment Laboratories agreed that wine isolated strains fermented cellobiose while strains isolated from beer did not ^[36].

Currently, research into how well Brettanomyces strains ferment the trisaccharide maltotriose has not been explored much by science, however, one study found that B. custersianus can ferment maltotriose. Another study found that all 7 strains of B. bruxellensis tested could ferment maltotriose, but not the trisaccharide raffinose. More investigation into this possibility is needed ^[37]^[18].

Just like in other yeast species, the temperature has a direct effect on the rate of fermentation for Brettanomyces. The optimal fermentation rate temperature range for Brettanomyces is between 22-32°C (77-90°F), however one study by Tyrawa et al. found that several strains of B. bruxellensis fermented at 30°C "smelled terrible" ^[36]. Fermentation rate is about half as slow at 20°C (68°F). Brettanomyces will still grow at temperatures as low as (and maybe lower than) 15°C (59°F) and will be much slower, however one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth and fermentation temperature range of 20-32°C. The growth rate at 15°C, while still slowly active, varies from strain to strain with some strains growing very poorly. Carbohydrates are consumed much slower, with cellobiose metabolizing ceasing for some strains (although phenol production stayed the same between 15°C and 22.5°C) ^[36]. At a temperature of 35°C (95°F), fermentation is greatly inhibited due to cell death. The primary byproducts of Brettanomyces fermentation, which are ethanol, acetic acid, and CO2, are produced both during growth but also during fermentation after growth has stopped. At the more optimal fermentation temperatures of 25-32°C, ethanol and acetic acid are produced faster from fermentation, but the amounts of ethanol and acetic acid produced from fermentation are not affected by temperature (i.e. higher temperatures do not produce more ethanol and acetic acid from the same amount of sugar, they are just produced faster at warmer temperatures because fermentation is faster) ^[38]. The warmer temperature ranges that are ideal for Brettanomyces fermentation rates and growth rates may still produce unfavorable flavors such as higher alcohols, however, this has not been analyzed as far as we know ^[39].

The below table is an example of the variety of sugar types that different strains/species of Brettanomyces banked at the National Collection of Yeast Cultures can ferment under semi-aerobic fermentation and aerobic growth (the semi-aerobic fermentation value is probably more useful for brewers since oxygen availability is limited during fermentation in normal brewing practices):

Species ^[34]	NCYC Num	Glucose (Semi-Aerobic/Aerobic)	Sucrose (Semi-Aerobic/Aerobic)	Maltose (Semi-Aerobic/Aerobic)	Cellobiose (Semi-Aerobic/Aerobic)	Lactose (Semi-Aerobic/Aerobic)	Soluble Starch (Semi-Aerobic/Aerobic)	Glycerol*	Ethanol*	Lactic Acid*	Succinic Acid*	Citric Acid*
D. anomala	NCYC 2	+/+	+/+	+/+	Weak or Latent/+	+/+	-/-	-	Weak or Latent	-	-	-
B. anomalus	NCYC 749	+/+	+/+	-/-	+/+	+/+	-/-	Weak or Latent	Latent	-	-	-
B. anomalus	NCYC 615	+/+	Weak or Latent/+	Weak or Latent/+	+/+	+/+	Unknown/Weak or Latent	+	Unknown	Unknown	Unknown	Unknown
D. anomala	NCYC 449	+/+	-/+	-/-	Unknown/+	Weak or Latent/+	Unknown/Unknown	Unknown	Unknown	Unknown	Unknown	Unknown
B. bruxellensis	NCYC 2818	+/+	+/+	+/+	-/-	-/-	-/-	-	+	-	-	-
D. bruxellensis	NCYC 2263	+/Weak or Latent	+/Weak or Latent	+/Weak or Latent	Weak or Latent/Weak or Latent	-/-	-/Weak or Latent	-	Weak or Latent	Weak or Latent	-	-
D. bruxellensis	NCYC 1559	+/+	Weak or Latent/+	Weak or Latent/-	Weak or Latent/+	-/-	-/-	+	+	-	-	-
D. bruxellensis	NCYC 823	+/+	+/+	+/+	Unknown/-	-/-	-/-	-	-	-	-	Unknown
D. bruxellensis	NCYC 395	+/+	+/+	+/+	-/Unknown	-/-	Unknown/Unknown	Unknown	Unknown	Unknown	Unknown	Unknown
B. bruxellensis	NCYC 370	+/+	+/+	Weak or Latent/+	Unknown/-	Unknown/-	Unknown/Unknown	Unknown	Unknown	Unknown	Unknown	Unknown
B. bruxellensis	NCYC 362	+/+	Weak or Latent/+	Weak or Latent/+	Unknown/-	-/-	Unknown/Unknown	Unknown	Unknown	Unknown	Unknown	Unknown
B. naardenensis	NCYC 3450	Weak or Latent/+	-/ Unknown	-/-	-/+	-/-	-/-	-	Weak or Latent	Weak or Latent	-	-
B. naardenensis	NCYC 3015	+/+	-/-	-/Weak or Latent	-/+	-/-	-/+	-	+	+	-	-
B. naardenensis	NCYC 924	+/+	-/-	-/+	Unknown/+	-/-	-/Weak or Latent	-	+	Weak or Latent	-	-
B. naardenensis	NCYC 899	Weak or Latent/+	-/-	-/Weak or Latent	Unknown/Weak or Latent	-/-	-/-	-	Weak or Latent	Weak or Latent	-	Unknown
B. naardenensis	NCYC 813	Weak or Latent/+	-/-	-/+	Unknown/+	-/+	-/-	-/Unknown	+	Weak or Latent	+	Unknown

* Measured only under aerobic utilization and growth because these compounds can only be metabolized under aerobic conditions ^[4].

Glycosides and Beta-Glucosidase Activity

Glycosides are flavorless compounds often found in plants/fruits that are composed of a molecule (often a flavor active compound) bound to a sugar molecule. The glycosidic bond can be broken, releasing the sugar molecule and the potential flavor active compound. These bonds can be broken with exposure to acid, as well as specific enzymes (beta-glucosidase) which can be added synthetically or produced naturally by some microorganisms, including some strains of Brettanomyces that have beta-glucosidase enzyme activity (mostly B. anomalus strains) ^[40]. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic ^[41]. It is speculated that flavor compounds from hops can also be released from glycosides ^[42], however, at least one study has shown no significant difference in a blind taste test between hopped beer exposed to the beta-glucosidase enzymes and hopped beer that was not exposed to the enzyme ^[43].

See the Glycosides page for more details.

Nitrogen Metabolism

Other than sugars, nitrogen is an essential nutrient for yeast and generally occurs in wort in the form of amino acids ^[44]. Brettanomyces can survive in environments that are very low in nitrogen. While nitrogen usage for S. cerevisiae is well understood, the general utilization of nitrogen by Brettanomyces and its preferred sources for nitrogen under the stressful conditions of fully fermented beer and wine are not yet well known. However, it is known that Brettanomyces can use a wide range of sources for nitrogen, and its requirements for nitrogen as a nutrient are extremely low when oxygen is available. When oxygen is not present, nitrogen is required for the survival and growth of Brettanomyces. Sources of nitrogen include amino acids such as lysine, histidine, arginine, asparagine, aspartic cid, glutamic acid, and alanine ^[4]. Some strains of Brettanomyces can metabolize other nitrogen sources, such as the amino acids proline and arginine ^[18]. Ammonium nitrates may also be utilized by some strains of B. bruxellensis. Although studies have been contradictory and some have not documented whether conditions were aerobic or anaerobic (these contradictions might also be due to strain differences between the B. bruxellensis strains that were used in different studies), it appears as though some strains of B. bruxellensis might be able to take advantage of trace amount of amino acids that S. cerevisiae does not use during fermentation, and nitrates and nitrites that S. cerevisiae is not able to consume, as well as amino acids from yeast autolysis (proline, leucine, tryptophan, and gamma aminobutyric acid) ^[4]. Other compounds from Saccharomyces autolysis may also be used by Brettanomyces, such as glucose, fatty acids, nucleotides, polysaccharides, polypeptides, and other proteins ^[45]^[46]. The role that oxygen plays in the ability of B. bruxellensis to uptake nitrogen from various sources might be an important one, and something that should be examined in science going forward ^[4].

Secondary Metabolites

Secondary metabolites are compounds that are not essential to the life of an organism ^[47]. Brettanomyces will use a range of secondary metabolites to produce many of the fruity and funky esters, phenols, and acids that this genus of yeast has become known for. Brettanomyces has also been observed anecdotally to produce thin beer when fermented on its own, and this has at least partially been attributed to the lack of glycerol production by Brettanomyces. The lack of glycerol production has been attributed to a genetic predisposal to prefer pyruvate production over glycerol production during fermentation, and it has been speculated that this gives Brettanomyces an adaptive advantage ^[48]^[22]. The major secondary metabolites of B. bruxellensis fermentation have been identified in one study as the ethyl phenols (4EP and 4EG), the alcohols isoamyl alcohol, 2-methyl-butanol, 2-ethylhexanol, phenethyl alcohol, and an ester ethyl 2-methyl butyrate. Many other compounds are considered minor secondary metabolites and are produced in varying degrees or not at all based on the strain of Brettanomyces, but may still be produced in high enough concentrations to contribute to the flavor and/or aroma in beers fermented with Brettanomyces. The types and amounts of flavor compounds produced by Brettanomyces cover a wide spectrum, and many factors such as species/strain, amino acid precursors, the presence of oxygen, and other nutrients, play a large role in the production of these compounds. In one study on Brettanomyces in wine, some strains rated as being perceived positively if the strains metabolized certain compounds slower and produced other compounds slower, indicating that the age of the fermented beverage also plays a large role in how beverages fermented with Brettanomyces are perceived ^[6]. Some strains of B. bruxellensis also produce the amines cadaverine, hexylamine, phenylethylamine, putrescine and spermidine, under wine-model conditions ^[8].

Ester Production

Brettanomyces is capable of synthesizing several ethyl esters from ethanol and fatty acids. Among the most prolific of these are ethyl acetate (synthesized from ethanol and acetic acid), ethyl lactate (synthesized from ethanol and lactic acid), phenethyl acetate, ethyl caproate, ethyl caprylate, ethyl deconoate ^[36], along with the hydrolysis (breakdown) of isoamyl acetate. During non-mixed fermentations where lactic acid is minimal to none, insignificant amounts of ethyl lactate esters are produced, whereas ethyl caprylate and ethyl caproate have a general increase. With the addition of lactic acid, ethyl lactate levels are greatly increased although may still not reach the flavor threshold level of 250 mg/L (strain dependent), and ethyl acetate is generally slightly increased. The amounts of esters produced vary widely based on species and strain ^[49]. A similar but slower evolution of esters has been seen in a long-term study on examining how Belgian lambic from Cantillon ages in bottles. The study found that lactic acid (produced by lactic acid bacteria) and ethyl lactate increased as bottles aged, while ethyl decanoate and isoamyl acetate decreased, all presumably from Brettanomyces metabolism over time ^[50].

Ester production peaks towards the end of growth and is influenced by temperature, aeration/agitation, and pH. Spaepen and Verachtert found in one study that the optimal temperature for growth and thus ester production was 28°C (77°F), although they did not test higher temperatures. This study also found that continuously shaken samples produced relatively fewer esters, as well as samples that were not exposed to oxygen at all. The highest ester production was found under conditions of limited oxygen supply, no agitation, held at a temperature of 28°C (77°F), and young cells produced more esters than older cells. It also found that esterase activity (esterase is the enzyme that facilitates ester production and destruction) increases as pH rises until a pH of 7.6 is reached, after which it begins to decline again. It was shown that the ester formation/degradation was indeed caused by enzymatic activity of any Brettanomyces species/strain, and not caused by chemical reactions or from Saccharomyces or Kloeckera activity ^[51]. Another study by Tyrawa et al. found that all strains of B. bruxellensis tested produced above threshold levels of ethyl caproate, ethyl caprylate, and ethyl deconoate esters at 15°C versus 22.5°C, but for some strains the higher fermentation temperature of 22.5°C produced significantly more of these esters than the lower 15°C temperature (other strains produced similar levels of esters at both temperatures, although they fermented slower at 15°C) ^[36].

Pitching rate of Brettanomyces may have a slight effect on ester production levels, but the differences caused by pitching rate probably do not have a significant impact on the sensory character of the beer ^[52]. Brettanomyces produces higher levels of esters when fermented without competition from S. cerevisiae, and this correlates with higher Brettanomyces cell growth when not in competition with S. cerevisiae (see 100% Brettanomyces Fermentation) ^[53]. The aromatic amino acids phenylalanine, tryptophan, and tyrosine have been associated with higher ester formation ^[6].

Esters are also broken down via a process called hydrolysis. Hydrolysis breaks the esters down using the same esterase enzyme within the Brettanomyces cells that are used to create esters. In general, all acetate based esters, except for phenethyl acetate and methyl acetate, are broken down faster than non-acetate esters by Brettanomyces. In lambic brewing, sometime after the primary fermentation finishes, Pediococcus begins to produce lactic acid. The formation of lactic acid by Pediococcus coincides with the appearance and growth of Brettanomyces, which produces more acetic acid. After another 2-3 months, the ester content of the lambic beer changes and reaches an equilibrium. Ethyl acetate and ethyl lactate are greatly increased, while isoamyl acetate is greatly decreased, reaching an equilibrium of these esters. Given a static amount of acetic acid, Brettanomyces reaches an equilibrium of ethyl acetate within 24 hours, while ethyl lactate equilibrium takes longer and is much more complex. In lambic, the majority of ester production and breakdown occurs within 1-3 months after lactic acid production by Pediococcus begins, and at a pH of around 3.5 and a temperature of around 15°C or less ^[51]. Pitching rate of Brettanomyces has an effect on the breakdown of isoamyl acetate with higher pitching rates breaking down this ester at a faster rate ^[52]. As far as we are aware, ethyl acetate is not metabolized further by Brettanomyces, and the level of ethyl acetate will not be hydrolized over time (although levels can continue to increase over time with more oxygen oxposure, since oxygen exposure encourages acetic acid synthesis by Brettanomyces and acetic acid bacteria, and acetic acid and ethanol are then metabolized into ethyl acetate by Brettanomyces).

Phenol Production

Phenols such as 4-vinylphenol (4VP, barnyard, medicinal) and 4-vinylguaiacol (4-VG, clove) can be produced in beer by the decarboxylation of hydroxycinnamic acids (HCAs), which are found in malt. While both Saccharomyces (only by "phenolic off flavor positive/POF+" strains) and Brettanomyces strains have varying capabilities based on strain of converting hydroxycinnamic acids to their vinyl derivatives ^[104], Brettanomyces is also able to reduce these vinyl phenol derivatives to ethyl phenol derivatives. Phenolic acid decarboxylase (PAD) is the enzyme that converts the HCAs into vinyl phenols. Vinyl reductase (VA) is the enzyme that reduces vinyl phenols to ethyl phenols ^[105].

These vinyl derivatives have similar tastes to the ethyl derivatives but have lower flavor thresholds. Levels of all compounds produced vary depending on species and strain of Brettanomyces. Although the production of ethyl phenols has been identified to occur higher in substrates with more available sugars, and this has also correlated with higher growth ^[106], some data supports that pitching rate does not have an effect on how much phenol content is produced by Brettanomyces^[52]. Additionally, Curtin et al. (2013) showed that while both cell growth and attenuation was inhibited in anaerobic conditions in wine, phenol production was not (in fact, the phenol production was inhibited by aerobic conditions). They also showed that each of the three strains of B. bruxellensis tested all produced the same amount of phenols, while other flavor compounds such as esters were produced at different levels by each of the strains ^[107]. Perhaps growth itself is not as much of a factor in producing phenols, but having sugars available for metabolism is. This contradicts the somewhat popular belief that under-pitching Brettanomyces produces more "funky" flavors. Additionally, perhaps some strains are perceived as "funkier" than others due to less ester production and more fatty acid production (isobutyric acid, for example), rather than more phenol production.

Another study by Tyrawa et al. found that fermentation temperature also did not have a significant effect on phenol production in 9 strains of B. bruxellensis. Given the same wort composition, strains of B. bruxellensis produced similar levels of phenols at both 15°C and 22.5°C. The ester production was affected by this temperature difference in some strains but not others (see Esters above). Assuming that phenols contribute the "funky" flavor characteristics, this suggests that perhaps a lower balance of esters to phenols produces a more "funky" tasting beer more so than a beer with more phenol content. If so, a lower fermentation temperature may be one way to emphasize phenols over fruity esters ^[36].

It has been hypothesized that the production and destruction of various phenols by Brettanomyces is connected with the redox balance, however, this has not been demonstrated. Ethyl phenols have been shown to be highly attractive to fruit flies, and it has also been proposed that these aromatics allow Brettanomyces to travel from food source to food source and by doing so increasing its chances of survival in the wild ^[108]^[4]. Phenols have been shown to have positive effects on decreasing protein glycation, a complication associated with type 2 diabetes ^[109].

Acid Production

In the presence of oxygen, Brettanomyces strains produce acetic acid as a byproduct of glucose fermentation. This is thought to be a defensive tactic against competing microorganisms (e.g. Brettanomyces has been shown to produce more acetic acid when co-fermented with S. cerevisiae, and S. cerevisiae has been shown to have less viability over time in the presence of acetic acid and ethanol) ^[53]. Depending on the brewer's palate and the degree of acetic production, this can be a desirable or undesirable trait. The degree of acetic acid production varies among different Brettanomyces strains and is limited by limiting oxygen exposure. Acetic acid produced by Brettanomyces is also used in the synthesis of acetate esters such as ethyl acetate, perhaps as a mechanism to protect itself after hindering other microbes via the acetic acid precursor. Brettanomyces has been shown to produce enough fatty acids in anaerobic fermentation to drop the pH to 4.0, which can also be esterified (see the ester table above) ^[61]. Many of these acids can have an unpleasant rancid odor and/or taste, which may be noticeable in young Brettanomyces beers before these acids are esterified.

Michael Lentz and Chad Harris tested whether or not the hydroxycinnamic acids (HCAs) inhibit the growth of Brettanomyces. They found that high levels of hydroxycinnamic acids (HCAs), which includes ferulic acid, p-coumaric acid, and caffeic acid, do inhibit the growth of Brettanomyces. Ferulic acid is the strongest inhibitor of these three HCAs with most strains tested not being able to grow in wort that contained 12 mM (millimolar) of ferulic acid. Caffeic acid was generally shown to be the weakest inhibitor of the three HCAs tested. Levels of 25 mM p-coumaric acid inhibited the growth of all strains tested, and levels of 30 mM of caffeic acid inhibited all strains tested. The ability of HCAs to inhibit growth is different from strain to strain of Brettanomyces. Inhibition does not appear to be species dependent. Some strains display a lag time and grow more slowly in the presence of high amounts of HCA's, but still eventually achieve maximum growth compared to if they were grown without exposure to HCAs, while others lag and then stop growing before reaching maximum growth ^[104].

The amount of HCAs varies widely from plant to plant, and the amount that is found in must or wort also varies on how the raw ingredients are treated. These measurements are generally not a consideration for maltsters or grape growers ^[104]. The one exception to this is the ferulic acid rest that German brewers have used to create more clove-like flavors in certain beer styles.

Acids Produced	Precursors	Notes
Acetic Acid (Vinegar, hard boiled egg)	Oxygen	Increased production with higher levels of oxygen exposure ^[61].
Butyric Acid (Vomit, bile)	Associated with supplements of phenylalanine or tyrosine ^[6]	Fatty acid.
Capric acid (Barnyard animal odor/taste) ^[61]^[6]		Fatty acid. Also found in milk, coconut oil, and seed oils ^[127]. Also referred to as Decanoic acid ^[128].
Caproic acid (Fatty, cheesy, waxy, barnyardy) ^[61]^[6]		Fatty acid. Also known as hexanoic acid ^[129].
Caprylic acid (Rancid-like smell and taste ^[61]^[6]		Fatty acid. Also found in milk. Gives a waxy/oily mouthfeel. Flavor is more intense at low pH levels. Also called octanoic acid.^[130]
Heptanoic acid (sweaty, solvent, rotten, barnyard)	Associated with phenylalanine or tyrosine ^[61]^[6]	Fatty acid. Also known as enanthic acid ^[131].
Isovaleric Acid (Feety, parmesan) ^[132]^[133]	Leucine	Commonly described as a "spoilage" acid produced by Brettanomyces in wine, but also appears in beer.
Lauric acid (faint odor of bay oil or soap) ^[61]		Fatty acid. Also known as dodecanoic acid and dodecylic acid ^[134].
Nonanoic acid (Rancid odor) ^[61]		Fatty acid. Also known as pelargonic acid ^[135].
Undecanoic acid (coconut, balsam, oily, vanilla) ^[61]	Associated with tyrosine ^[6].	Fatty acid. Also known as undecylic acid ^[136].

Other Compounds

Compound Produced	Chemical Type	Precursors	Threshold	Notes
2-Methoxyphenethyl alcohol (white glue, plastic, mimeograph sheet)	Alcohol	Associated with ferulic acid ^[6]
2-Ethyl-1-hexanol (fake floral, chemical, fusel oil)	Alcohol	Associated with caffeic acid, ferulic acid, tyrosine, and phenylalanine		Produced by many strains of B. bruxellensis. Identified as a major product of B. bruxellensis ^[6].
2-Methyl-1-butanol (oxidized/canned fruit, plastic, sulfur)	Alcohol	Associated with ferulic acid, phenylalanine, and tyrosine ^[6].		Identified as a major product of B. bruxellensis ^[6].
Bisabolene (spicy, tropical, toasty, wood resin, minty)	Terpene	Associated with caffeic acid ^[6].
Decanol ^[6]	Alcohol			Also known as decyl alcohol ^[137].
Isoamyl alcohol ^[6]	Alcohol	3 Methylbutanal		One of several isomers of Amyl alcohol; also known as 3-methyl-1-butanol. It is a major higher chain alcohol produced in fermentation ^[138]. Commonly produced by many strains of B. bruxellensis. Identified as a major product of B. bruxellensis ^[6].
Nerilidol ^[6]	Terpene			Often found in bitter gourd and is a component of many essential oils. It is often used as a flavoring agent ^[139].
Nonanal (lemon furniture polish, air fresener, oily	Aldehyde	Associated with tyrosine ^[6]
Ocimene (sweet floral, hyacinth, jasmine)	Terpene	Associated with phenylalanine ^[6]
Octanol ^[6]	Alcohol			A colorless, slightly viscous liquid used as a defoaming or wetting agent, and is found naturally as a part of esters in some essential oils ^[140].
Phenethyl alcohol ^[6] (floral, dried rose ^[141])	Alcohol			Also known as phenethanol. Identified as a major product of B. bruxellensis ^[6].
Phenylacetaldehyde ^[6] (honey, floral rose, swaat, powdery, chocolate with a slight earthy nuance ^[142])	Aldehyde
Tetrahydropyridine (Cheerios®, mousy, urine, cracker biscuit, corn chips)	Ketone ^[143]	L-Lysine, ethanol, and oxygen	Varies	See the Tetrahydropyridine page for more details.

Commercial Cultures

Pure cultures. In cooperation with Funk Factory.

Bootleg Biology

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Unknown	Unknown	Unknown	Funk Weapon #1 BB0034A	This rare, and commercially unavailable yeast isolate, produces pungent horse blanket and fresh leather aromas. Perfect for breaking out the funk in farmhouse-style beers. This is the first release in the Dusty Bottoms Collection’s ongoing Funk Weapon Series of unique, rare Brettanomyces and Brett-like wild yeast cultures.	West Flanders, Belgium brewery specializing in funky, sour, mixed-fermentation beers.
Unknown	Unknown	Unknown	Funk Weapon #2 BB0035C	This rare, and commercially unavailable yeast isolate is perfect for 100% Brettanomyces fermentations, especially Brett IPAs. Amplifies citrus and tropical fruit-forward hop flavors and aromas into a punchy ripeness. Great for maintaining the nuance of hops in beer with greater keeping qualities than a Brewer’s Yeast fermentation. You can’t make every style of beer with one or two strains of brewer’s yeast, so why would you only use only one or two strains for your funky beers? This is the second release in the Dusty Bottoms Collection’s ongoing Funk Weapon Series of unique, rare Brettanomyces and Brett-like wild yeast cultures.	Family-owned brewery producing Gueuze in West Flanders, Belgium.
Unknown	Unknown	Unknown	Funk Weapon #3 BB0022	Funk Weapon #3 is a versatile culture that creates wildly different flavor and aroma profiles depending on the age of fermentation. Young fermentations produce mild musty funk and ripe tropical fruit, while older and bottle conditioned ferments show off unique flavors and aromas of strawberry, cherry and tropical candy. This commercially unavailable yeast isolate is ideal for 100% Brettanomyces fermentations or as a secondary strain along with a phenolic Brewer’s Yeast culture.	Dry-hopped, Unblended Lambic Produced by a Traditional Lambic Brewery in Brussels, Belgium.

Brewing Science Institute

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Brettanomyces bruxellensis	Medium intensity Brett character. Classic strain used in secondary fermentation for Belgian style beers and lambics.	Same as White Labs. Pro-Brewers only.
Claussenii	Dekkera anomala	B. claussenii	Brettanomyces clausenii	Low intensity Brett character.	Same as White Labs. Pro-Brewers only.
CMY1	Dekkera bruxellensis	B. bruxellensis CMY1	bsi		Chad Yakobson's mutation of BSI Drie. Pro-Brewers only.
Drie	Dekkera bruxellensis	B. drei	Brettanomyces bruxellensis var. Drei	Highly aromatic brett strain. Sourness takes extensive aging to produce	Isolate from Drie Fonteinen; Pro-Brewers only. Recommended fermentation levels to get the most character out of Drie include 75-77°F ^[144].
Lambicus	Dekkera bruxellensis	B. lambicus	Brettanomyces lambicus	High intensity Brett character. Know to produce the “horsey” aroma characteristic of Brettanomyces yeast. Classic strain used in secondary fermentation for Belgian style beers and lambics. Same as White Labs.	Pro-Brewers only.

East Coast Yeast

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Anomala	Dekkera anomala	B. intermedius	ECY-04	strong ester profile with some light funk and acidity	beer - Adelaide, Australia
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	ECY-05	funky with barnyard notes accompanied by some fruit	isolated from Belgian stout
Custersianus	Brettanomyces custersianus	B. custersianus	ECY-19	light fruit and hay	Bantu beer brewery, South Africa
Farmhouse	?	B. fantome	ECY-03b	Fruity and funky profile with some acidity gradually increasing over time. Aeration has more of a muted effect	Isolate from Fantome. May not be Brett as per Lance Shaner on MTF.
Naardenensis	Brettanomyces naardenensis	B. naardenensis	ECY-30	strawberry, honey, ripe fruit with a tart, citrusy acidity after 6mo of aging	Isolated from Dr. Pepper
Nanus	B. nanus (or Eeniella nana; see Taxonomy)	B. nanus	ECY-24	This species of Brettanomyces reveals a phenolic, spicy, saison-like profile with none to low esters. Some earthiness and tartness may develop over time. Slow to attenuate fully alone, may be best in fermenting with other yeast.	bottled beer - Kalmar, Sweden

Escarpment Laboratories

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Brett B	A classic Brettanomyces bruxellensis strain, typically used in secondary fermentations. Attenuation: 70-85% // Optimum Temp: 26ºC+ (80F+) // Alcohol tolerance: Medium-high // Flocculation: Low
Claussenii	Dekkera anomala	B. claussenii	Brett C	A strain of Brettanomyces clausenii, now understood to be among the Brettanomyces anomalus species. Minimal funk, tends to exhibit fruity pineapple or mango notes. Pairs well with fruit and/or hops. Recommended for secondary or co-fermentation as attenuation is variable. Attenuation: Highly Variable // Optimum Temp: 18-25ºC (64.4-77F) // Flocculation: Medium-low
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Brett D	This strain of Brettanomyces bruxellensis is a notoriously vigorous fermentor, suitable for primary fermentation of 100% Brett beers or secondary fermentation where some extra funk is desired. Attenuation: 80+% // Optimum Temp: 20-25ºC (68-77ºF) // Alcohol tolerance: 12%+ // Flocculation: Medium-low
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Brett Q	This strain typically completes primary fermentation within one month, and is also suitable for secondary aging of a wide range of beer styles where subtle Brett character is desired. Tasting notes include ripe strawberry, pear, apple, with underlying funk. Attenuation: 80%+ // Optimum Temp: 20-25ºC // Alcohol tolerance: High // Flocculation: Medium-low	Originally isolated from a barrel-aged sour beer from Quebec.

Imperial Organic Yeast

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
W15 Suburban Brett	Dekkera bruxellensis	B. bruxellensis ^[145]	W15	Suburban Brett is Brettanomyces yeast that works great as a secondary aging strain. It really shines when used in wood barrels and will produce complex and balanced aromas of sour cherry and dried fruit. It can also be used for as a primary strain for Brett only beers. Temp: 64-74F, 18-23C // Flocculation: Low // Attenuation: 75-80% ^[146]. MTF thread on experiences with this culture.

Inland Island Yeast Laboratories

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	INIS-901 Brettanomyces bruxellensis I	Isolated from a brewery in Brussels this particular Brettanomyces strain in known for producing aromatics reminiscent of horse, barnyard, sweat, and goat. It is highly attenuative and will take up to 6 months to fully finish fermentation. It is suggested that this strain be used with another primary fermentation strain. 90% + Attenuation. Low Flocculation. 60-75 F Temperature Range. High Alcohol Tolerance.	Brewery in Brussels
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	INISBC-902 Brettanomyces bruxellensis II	This strain is capable of fermenting a beer without the use of a Sacchromyces c.. This yeast produces a beer with a complex flavor profile, containing both fruity and sour notes. 85%+ Attenuation. Low Flocculation. 70-85 F Temperature Range. Medium-High Alcohol Tolerance.
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	INISBC-903 Brettanomyces bruxellensis III	Isolated from a small brewery just outside of Brussels. Produces an aromatic profile that is more mild than INISBC-901 with increased tropical fruitiness. Able to ferment a beer without added Saccharomyces c. Mixed with lactobacillus this strain will create a wonderful sour beer. 90% + Attenuation. Low Flocculation. 60-75 F Temperature Range. High Alcohol Tolerance.	Small Brewery just outside of Brussels
NA	NA	NA	INISBC-913 Brett Barrel Yeast III	Strain is able to ferment without added help from another Saccharomyces c. strain. Produces mild acidity and tropical fruit notes. Leaves the beer with very little mouthfeel. See recipe recommendations for fermentation additions to boost mouthfeel. 90% + Attenuation. Low Flocculation. 60-75 F Temperature Range. High Alcohol Tolerance.	Isolated from a famous american wild ale brewery.
Lambicus	Dekkera bruxellensis	B. lambicus	INISBC-920 Brettanomyces lambicus	Originally isolated from spontaneously fermenting beers, this strain will give your beer textbook Brett flavors like horse blanket and spice, but also fruity notes of pineapple. Fermentation will take well over a month to fully attenuate if used as a primary strain. 85%+ Attenuation. Low Flocculation. 70-85 F Temperature Range. Medium-High Alcohol Tolerance.
Claussenii	Dekkera anomala	B. claussenii	INISBC-930 Brettanomyces claussenii	Possibly the least “Bretty” of the Brettanomyces strains, Claussenii adds great fruity pineapple aroma without the traditional flavors that turn some off from Brett fermented beers. An excellent choice for adding a little funk to your barrel aged favorite. 85%+ Attenuation. Low Flocculation. 85 F+ Temperature Range. Medium-High Alcohol Tolerance.
NA	NA	NA	INIS-950 Brettanomyces "Copenhagen"	This unique yeast will add notes of barnyard and ripe fruit to beer.	Isolated from a craft brewery in Denmark ^[147].

GigaYeast

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Brussels Bruxellensis GB001	Produces classic Brett “Barnyard” characteristics plus some subtle fruity aroma and moderate acidity. Adds a tart complexity to any beer.
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Tart Cherry Brett GB002	Produces some Brett Barnyard funk plus stone fruit and cherry-like esters. This Strain also produces a moderate amount of acid that adds a tart complexity to the brew.
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Sweet Flemish Brett GB144	Produces a sweet, slightly fruity profile with just a hint of barnyard and spicy phenolics

Jasper Yeast LLC

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis "Chateaux"	JY87	JY87 is a Brettanomyces yeast isolated by jasperyeast LLC. Fast-growing, it has almost Saccharomyces-like doubling times. Shows great fermentation as primary strain in a variety of beers. Ferments fast to 60% attenuation, after which fermentation slows down and more flavor and aroma is produced. Strong pineapple and stonefruit aroma after prolonged fermentations (3-9 months). Great companion to beers that could use some funk, and complements hoppy beers perfectly. Flocculation is low, strain will form a pellicle when oxygen is present. Sequencing of ITS regions indicated Brettanomyces bruxellensis. Micrograph of JY87 cells coming soon.	West-Flanders, Belgium.

Omega Yeast Labs

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma
Claussenii	Dekkera anomala	B. anomala	Brettanomyces claussenii OYL-201	Contributes more Brett aroma than flavor. Fruity, pineapple like aroma. Flocculation: low, Attenuation: 70-85%, Temp: >85°F, Alcohol Toelrance: medium-high, compares to WLP645. Pro brewers only.
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Brettanomyces bruxellensis OYL-202	Medium intensity Brett character. Classic strain used in secondary fermentation for Belgian style beers and lambics. Flocculation: low, Attenuation: 70-85%, Temp: >85°F, Alcohol Tolerance: medium-high, compares to WLP650. Pro brewers only.
Lambicus	Dekkera bruxellensis	B. lambicus	Brettanomyces lambicus OYL-203	This strain has been described as producing horsey, smoky and spicy flavors. As the name suggests, this strain is found most often in Lambic style beers. Flocculation: low, Attenuation: 70-85%, Temp: >85°F, Alcohol Tolerance: medium-high, compares to WLP653. Pro brewers only.

RVA Yeast Labs

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	RVA 502	A medium-intensity Brettanomyces yeast strain. Will add a bit of funk when added during the secondary. Typically used in Belgian-style beers, especially lambic. A famous Trappist brewery produces its unique beer with this yeast during secondary fermentation.
Claussenii	Dekkera anomala	B. claussenii	RVA 501	A low-intensity strain. Contributions from this strain are mostly aromas of pineapple and fruit. This strain prefers higher temperatures (85º F), but will produce nice aroma and subtle flavor at normal ale fermentation termperatures (68-72º F).
Lambicus	Dekkera bruxellensis	B. lambicus	RVA 503	High-intensity “Brett” strain. Very spicey with “smoky” and “horseblanket” flavors and aromas. This strain is used mostly in Lambics and Flanders sour beers.
Unknown	Unknown	Unknown	RVA 804	Produces some amazing aromas of pears, and other fruit esters. We highly recommend this strain for Belgian Dubbels. This strain also makes a very nice cider. A highly flocculating, medium-high attenuating strain adds nice complexity to stouts, and Belgian Ales and Specialty Belgian Ales. Flocculation: Medium, Attenuation: 78-85%, Suggested Temp Range: 65-72°F, Alcohol Tolerance: 14%.	This strain originates from local fruit trees.

The Yeast Bay

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	TYB184	This strain is the first single strain Brettanomyces isolate we are releasing all on its own because it deserves that distinct honor. Isolate TYB184 is literally the 184th isolate of yeast/bacteria that has made it through primary fermentation trials, was assigned an isolate number and carried into larger scale fermentation evaluation since our conception in 2013. This isolate is attenuative, produces a moderate acidic-like character and an ester profile of lemon/pineapple. Another notable characteristic of this isolate is the mild barnyard character it produces that doesn't take over the profile; rather, it balances the ester profile. The unique character balance in this strain is what makes it well suited for use on its own, in both primary and secondary fermentation. Approximately 15 billion cells/vial. Temperature: 72 - 85 ºF. Attenuation: 82%-88%. Flocculation: Medium-Low	Isolated from a rustic farmhouse style beer produced in the Northeastern United States ^[148].
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	TYB207	This isolate exhibits good attenuation, and produces a moderate acidic-like character and an ester profile the combination of which produces a character reminiscent of sweet tarts. It's a fruity, funky tartness that's refreshing and crisp. This strain is well-suited for primary and secondary fermentation. Approximately 15 billion cells/vial. Temperature: 70 - 82 ºF , Attenuation: 80%-82%, Flocculation: Medium-Low.	Isolated from a Belgian-inspired brewery in the Northeastern United States ^[149].
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	TYB261	This Brettanomyces isolate exhibits a mild tartness and soft funk with a solid backbone of tropical fruit esters (papaya, guava, pineapple, guinep). It's great primary fermenter, but really shines in secondary fermentation following up a primary fermentation that produces a lot of flavor compounds. This strain is a true flavor modulator, and the more raw material it has to work with the greater the complexity that will be achieved in the finished beer. Sequencing results revealed it's Brettanomyces bruxellensis. This strain will produce a massive, thick krausen, so be sure to use a blowoff tube or reserve plenty of fermentor headspace! Isolated from a unique mixed fermentation beer produced in the Midwestern United States ^[150].

White Labs

See the public Yeastman database for exact cell counts for specific lot numbers.
See attenation rates based on pitch rate from this White Labs data sheet.

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	WLP650	Barnyard. Prominent Leathery aroma and flavor. Low levels of supporting tropical fruit in the aroma and flavor. ^[151]	Not the same as WY's Brux. Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL ^[152].
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis Trois Vrai	WLP648	Pear. Overly ripe stone fruit and pineapple, with hints of citrus in the aroma. Pineapple, overly ripe tropical stone fruit, lemon and lime rind flavor. Little to no "funk". Less complex overall compared to other Brettanomyces cultures from WL ^[151].	The vrai (true, in French) Brettanomyces bruxellensis Trois. The infamous strain used for all-Brettanomyces fermentations, has a robust, complex sour character with aromas of pear. Best used as a primary fermentation strain. May be the same as BSI Drie? Profile is very similar to BSI Drie ^[153] ^[154]. Ale of the Riverwards sensory analysis suggests they may be different strains. Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL ^[152]. Bryan of Sui Generis blog and Devin Henry found that this culture contains two closely related B. bruxellensis strains with very different flavor profiles ^[12]^[155].
Claussenii	Dekkera anomala	B. claussenii	WLP645	Fruity, pineapple. Wine grape-like aroma, with light wood-like, floral, and citrus aromas. More fruit forward in the flavor, clean aftertaste with little to no "funk" ^[151].	Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL ^[152]. See also this MTF thread and this MTF thread which discuss the purity of this culture, and references Yakobson's data that indicates that it does not attenuate wort efficiently when purely isolated.
Lambicus	Dekkera bruxellensis	B. lambicus	WLP653	Horsey, Smoky, Spicy. High amount of ripe pineapple and overly ripe stone fruit in the aroma and flavor, with mild levels of blue cheese, leather, and spicy phenol in the flavor ^[151].	Different from WY's "lambicus". Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL ^[152].

Wyeast

Common Name	Species Name	Synonym (Strain) Name	Lab/Package	Flavor/Aroma	Source Note
Anomalus	Dekkera anomala	B. anomalus	Wyeast 5110		bottled stout - Burton on Trent, England. Discontinued in 2007 due to being misclassified ^[156]. Cell count: 7.5 x 10⁸ cells/mL ^[157].
Bruxellensis	Dekkera bruxellensis	B. bruxellensis	Wyeast 5112	"sweaty horse blanket"	Not the same as WL's Brux. Cell count: 7.5 x 10⁸ cells/mL ^[157].
Claussenii	Dekkera anomala	B. claussenii	Wyeast 5151	Notes of tropical fruit, pineapple and, to a lesser extent, peach and blueberry round out a classic Brett profile. Produces “horse blanket,” leathery, and smoky character, but at lower level than other Brett strains. Can be used as the primary strain for fermenting, but is often used after a primary fermentation with an S. cerevisiae strain, and in blends to produce sour beers. It is highly attenuative, given proper time to fully ferment out, and is known to create a pellicle during fermentation.	Private collection for Spring 2015/Summer 2016. Cell count: 7.5 x 10⁸ cells/mL ^[157].
Lambicus	Dekkera bruxellensis	B. lambicus	Wyeast 5526	Pie-cherry	Different from WL's "lambicus". Cell count: 7.5 x 10⁸ cells/mL ^[157].

Smaller Labs

Mfg	Package	Notes
BKYeast	Brett X1	Suspected Brettanomyces Anomalus
BKYeast	Brett C1	Isolate from Cantillon Iris
BKYeast	Brett C2	Isolate from Cantillon Iris
BKYeast	Brett C3	Isolate from Cantillon Iris
Blackwell Brewery	BWY-001 Berliner Brett I	This Brettanomyces yeast was isolated from a Willner-Brauerei-Berlin Berliner Weisse. A Brettanomyces yeast strain that develops a rather subtle flavour profile but leads to a super dry finish. The perfect yeast to create a classical interpretation of a Brettanomyces bottle conditioned Berliner Weisse.
Craft Cultures	CCYL410 Razz Brettanomyces™	MICHIGAN INDIGENOUS - The Razz Brettanomyces™ is the first in a series of indigenous Brettanomyces strains. This yeast was isolated from a wild razz berry in Michigan's Upper Peninsula along the shore of the Portage Lake near Houghton Michigan. Great for use in secondary fermentation for Belgian style beers and lambics.
DCYeast	DCY01
Saccharolicious	Brett I	Brettanomyces yeast from a Walloon Trappist brewery that gives an earthy aroma to the beer. Recommended for secondary fermentation after primary fermentation with Trappist O.
Saccharolicious	Brett II	Fruity Brettanomyces yeast strain with an aroma that reminds of French cider. originates from Brasserie à Vapeur in Pipaix, Belgium, and was isolated from a bottle of Cochonne.

Brett Blends (Brett only)

Manufacturer	Package	Notes
East Coast Yeast	ECY34 Dirty Dozen Brett Blend	Twelve (12) different isolates of Brettanomyces exhibiting high production of barnyard "funk" and esters. Dryness, ripe fruit, and acidity will be encountered over a period of months and over time (>1 yr), may display gueuze-like qualities in complexity. Contains various isolates from lambic-producers, B. bruxellensis, B. anomalus, B. lambicus, and B. naardenensis. For those who want the most from Brett yeast, whether a 100% Brett fermentation is desired or adding to secondary aging projects. Suggested fermentation temperature: 60-74 F. Attenuation high. See this MTF thread for fermentation tips for 100% and mixed fermentations.
GigaYeast	Brux Blend (GB156)	A blend of Brettanomyces yeast that produces stone fruit esters and a hint of barnyard. Creates a moderate amount of acid that adds a tart complexity to the brew.
Omega Yeast Labs	All The Bretts OYL-218	This will be an evolving blend comprised of nearly every Brettanomyces strain in our collection (inaugural release will contain 12 strains). When used in secondary, expect high attenuation and a fruity and funky complexity developing over time. Attenuation: 85+%; Flocculation: low; Temperature: 68F-85F. Homebrew pitch contains ~70 billion cells ^[158].
The Yeast Bay	Beersel	Not overly funky but there is a sweaty note hanging behind lemon and citrus fruits, nice blend of subtle funk and citrus/fruit. All strains were identified as B. bruxellensis ^[159]. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer ^[160].
The Yeast Bay	Brussels	Similar to Beersel but with more funk in aroma and less fruit, complex barnyard character. All strains were identified as B. bruxellensis ^[159]. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer ^[160].
The Yeast Bay	Lochristi	Smells of Iris C2, probably the same, subtle blend with some delicate fruit, strawberry. All strains were identified as B. bruxellensis ^[159]. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer ^[160].
The Yeast Bay	Amalgamation Brett Super Blend	6 Brett blend to create a dry beer with a bright and complex fruit-forward flavor and aroma, accompanied by some funk. All strains were identified as B. bruxellensis ^[159]. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer ^[160].

Using Brettanomyces

Primary versus Secondary Fermentation

Brettanomyces can be pitched into a beer at many points in the beer's fermentation life cycle. If used as the primary fermenter, the beer that is produced is often fruit forward and not very funky. A large cell count will be needed (somewhere between an ale and lager pitching rate). See the 100% Brettanomyces Fermentation page for more information. If pitched into a beer that has already been fermented by Saccharomyces, a wider range of flavors including the funkier flavors can be produced (see the Brettanomyces Metabolism section above), although Brettanomyces can produce phenols on its own (de novo) without phenolic precursors produced from Saccharomyces. A small cell count of Brettanomyces is plenty for creating these flavors, and normally a starter is not necessary. See the Mixed Fermentation, Brettanomyces and Saccharomyces Co-fermentation, and Brettanomyces secondary fermentation experiment pages for more information on using Brettanomyces in secondary.

Starter Information

When pitching just Brettanomyces from a commercial pure or blended culture and no other microbes, it is recommended to make a starter for the culture. If the Brettanomyces is being pitched into secondary, no starter is necessary unless the brewer suspects that the Brettanomyces has lost a lot of viability due to age, heat exposure, etc., or prefers higher cell count pitches (current information suggests that there is no significant flavor difference between high and low pitching rates in secondary pitches of Brettanomyces; see Brettanomyces secondary fermentation experiment). Brettanomyces growth is inhibited by the concurrent growth of S. cerevisiae under anaerobic conditions. In aerobic conditions, oxygen appears to help Brettanomyces out-compete S. cerevisiae. The presence of lactic acid bacteria does not greatly effect Brettanomyces growth ^[53]^[161]. Therefore, when making starters for mixed cultures of Brettanomyces and Saccharomyces, the brewer can favor Saccharomyces by limiting oxygen, or favor Brettanomyces by introducing oxygen during growth. Data from Thomas Hübbe and Mark Trent support that the initial pitching rate doesn't have a great effect on the final cell count in pure Brettanomyces starters or beer, indicating that Brettanomyces is fairly forgiving in regards to small initial cell counts ^[53]^[162].

Just like in other yeast species, temperature has a direct effect on the rate of growth for Brettanomyces. The optimal growth rate temperature range for Brettanomyces is between 25-32°C (77-90°F). Growth is about half as slow at 20°C (68°F). Brettanomyces will still grow at temperatures as low as (and maybe lower than) 15°C (59°F) and will be much slower, however one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth temperature range of 20-32°C. At a temperature of 35°C (95°F), both growth and viability over time are greatly inhibited ^[38].

For information on mixed culture starters, see Mixed Culture Starters.

Two Approaches to Starters

There are generally two approaches to handling Brettanomyces starters. The first is to use a stir plate set to a medium-high RPM with tin foil on top of the flask for 7-8 days, cold crash for a few days, and then decant the beer before pitching the sedimented yeast. The second approach is to use an orbital shaker set to 80 RPM to create a semi-aerobic environment (this means that the oxygen levels are low, but also not non-existent) for 7-8 days as described in The Brettanomyces project ^[163], cold crashing can be skipped, and the entire starter is pitched into the wort. An alternative to the second approach is to use a stir plate on a very low setting so that only a very small "dimple" of a vortex is formed ^[164]. If a stir plate is not available, give the starter an initial dosage of pure O2, and then cover it with foil so that oxygen can slowly diffuse into the starter, and gently agitate as often as possible ^[165].

Oxygen levels are an important factor to consider when deciding which of the above two methods to use for a Brettanomyces starter. Brettanomyces creates acetic acid in the presence of oxygen, potentially leading to higher levels of ethyl acetate, which is considered an off flavor in higher amounts. As the amount of oxygen increases, cell growth increases, but so does acetic acid production. The amount of acetic acid produced is species/strain dependent, so some strains may benefit from more aeration without having the negative effect of creating too much acetic acid. Other strains may need a less aerobic starter (semi-aerobic) in order to produce the highest cell count with minimal acetic acid ^[166]^[167]^[168]. In addition to acetic acid production, it has been observed that some Brettanomyces strains grown under aerobic conditions continue to produce THP when transferred to anaerobic conditions. See THP for details.

This presents a sort of "catch 22" when growing Brettanomyces in a starter. The brewer must weigh the pros and cons of how much aeration to provide. If the Brettanomyces is going to be used in a 100% Brettanomyces Fermentation, for example, then a stir plate may be the best choice. If the Brettanomyces is instead being pitched in secondary with the intention of long aging, then having a high cell count isn't as necessary and the risk of adding more acetic acid/ethyl acetate to an aging beer is greater. If a lot of acetic acid is produced during the starter, it is advised to cold crash and decant the starter. Brettanomyces can have a difficult time flocculating and settling out, even when cold crashed. The brewer may need to allow a few days for the cells to fully sediment ^[169]. Additionally, Brettanomyces that is cold crashed may be slower to begin fermentation. If the brewer believes that the amount of acetic acid produced was insignificant, then cold crashing can be skipped and the entire starter can be pitched.

Although more experiments are probably needed, agitation is believed to be an important factor for any species of microbe (yeast and bacteria). Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth ^[170].

Maintaining a temperature of 77°-86°F/25°-30°C results in faster growth than lower temperatures and is recommended ^[171]. Brettanomyces cell growth typically takes about 7-8 days to reach it's maximum growth ^[172], however some strains may grow at faster rates and finish in 3-4 days ^[173]. When the starter turns a rich creamy color, it should be done within a few hours after this visual indication occurs ^[171]. Each step of a starter for Brett should be 7-8 days (or 3-4 days for faster growing strains).

For more information regarding aeration and agitation effects on Brettanomyces growth, see Mark Trent's Brettanomyces Propagation Experiment.

Pitching Rate Calculators

Current yeast pitching calculators for brewers are not adequate for determining Brettanomyces pitching rates based on starter volume size because the maximum cell density of Brettanomyces per mL of wort is 3 to 6 times the cell density of Saccharomyces. For example, a given Saccharomyces strain may reach a cell density of 130 million cells per mL in a 1.040 wort (different Saccharomyces strains can have different cell densities as well, although they are a lot lower than Brettanomyces overall). Different Brettanomyces strain cell densities have been reported to be 600 to 885 million cells per mL in 1.040 wort depending on the species/strain ^[172]^[174]. Since yeast calculators are based on S. cerevisiae or S. pastorianus cell density, using one of these tools for Brettanomyces starters will create an unexpectedly high cell count in reality. There is not currently enough data to accurately determine starter volumes for Brettanomyces, particularly because each strain and species have a different maximum cell density per mL of wort. However, pitching around 500-600 mL of a Brettanomyces starter for 5 gallons of 1.060 SG wort will achieve a pitching rate that is similar to lager yeast pitching rates, which has been recommended for 100% Brettanomyces Fermentation. Omega Yeast Labs is currently working on a project to create a more accurate Brettanomyces pitching rate calculator (it will also contain pitching rate calculations for specific strains of Saccharomyces, which is something that current yeast pitching calculators do not include) ^[174].

Given this information, many brewers historically have been using the lager pitching rate settings in online yeast pitching calculators for Brettanomyces starters (around 2000 mL, for example). Effectively, this means they have been pitching around 4 to 5 times the amount of Brettanomyces cells that they thought they were pitching. However, if this very high pitching rate is giving good results for brewers, it should continue to be used. Exploration of Brettanomyces pitching rates for 100% Brett fermentations is something to be desired once we know what our pitching rates actually are, and many brewers have been pitching 4-5 times the pitching rate for lagers if they use an online yeast pitching rate calculator instead of counting the cells under a microscope.

MYPG Growth Substrate

For yeast laboratories, "Malt Yeast Peptone Glucose" growth substrate has been shown to be a better substrate than wort for initially growing Brettanomyces from a plate or slant. When grown in wort, Brettanomyces will often go through a 24 hour lag phase, a growth phase, another lag phase, and a second growth phase (all within 7-8 days). When grown in MYPG substrate, there is only a single growth phase and no lag phase, which has been reported by Yakobson to produce a larger cell count in the same amount of time ^[175]. Cells grown in MYPG also are better adapted to grow in wort ^[176]. Practical instructions for making this substrate can be found on Jason Rodriguez's blog, "Brew Science - Homebrew Blog". Unfortunately, growing Brettanomyces pitches in MYPG for breweries isn't very practical due to needing almost 4 times the amount of MYPG versus wort to get the same pitching rate. In a brewery or homebrewery, using wort for Brettanomyces starters is more practical ^[177].

Cell Counting

The use of methylene blue, although popular in breweries, has been shown to be inaccurate when counting cells of Brettanomyces. Trypan blue staining has been shown to give more accurate cell counting results ^[53]^[178].

Example of a Home Lab Orbital Shaker

Mark Trent's shaker platform (obtained from a used equipment outlet in Gilroy, CA called "Outback Equipment" ) used to create a semi-aerobic environment for Brettanomyces. Mark built an insulated box for it, and added temperature control. He can propagate up to 7 liters. This is running at 80 RPM as described in The Brettanomyces project ^[163]^[179].

Storing Brett

Major yeast labs will often store yeast in a -80°C laboratory freezer in a media/glycerol solution, however this option is generally not practical for brewers ^[180]. The next best option for long term storage of Brettanomyces is freezing with 10% glycerol in a home freezer, however the effects of storing yeast at such a high and often variable temperature have not been evaluated scientifically. Traditionally Saccharomyces yeast has been stored on slants held in a refrigerator and can provide storage for a few months up to 2+ years, depending on the type of slant used (using mineral oil in slants has been shown to extend the life of stored Saccharomyces). Homebrewers however have reported poor survival of Brettanomyces on slants. A study underway by a MTF member is showing promising results by buffering the slant media. In this study, Brettanomyces has stored well for up to100 days on the buffered media. Another evaluation will be done after 6 months in storage. For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, see Bryan's video on Sui Generis Brewing (requires a pressure cooker). Agar plates are the least best solution and have been observed anecdotally to reduce viability of Brettanomyces over a few months ^[181]^[182].

Perhaps the best method for storing Brettanomyces long term is in sterilized (autoclaved or pressure cooked) wort or MYPG. Although not as ideal as freezing with glycerol at -80°C, this is the most practical way to store Brettanomyces for brewers without a lab freezer. Regarding temperature, it has been shown that cold storage for as long as a month is better than room temperature. However, after one month Brettanomyces appears to be more viable when stored at room temperature. More data is required before assuming this is the case with all strains of Brettanomyces. Chad Yakobson noted that after storing Brettanomyces in a refrigerated environment (we don't know how Chad was storing the Brettanomyces cultures when he observed this, for example on agar plates or slants or something else.), after 6 months the Brettanomyces would die. If Brettanomyces is stored cold, it will be very sluggish and slow to start fermentation. Making a starter is highly recommended if the Brettanomyces culture has been stored cold ^[183].

In order to explore Yakobson's anecdotal observations in a more controlled manner, Mark Trent performed an experiment on storing one strain of Brettanomyces in wort, MYPG, buffered wort (buffered to prevent a drop in pH), and buffered MYPG, and compared storage of the Brettanomyces in each of the storage solutions at room temperature versus cold temperatures for 100 days. This single Brettanomyces strain survived best in unbuffered MYPG at room temperature, and second best in unbuffered wort at room temperature, and survived less in cold storage conditions for all media. See the Brettanomyces Storage Survival Experiment for more details. Therefore, when storing Brettanomyces for one month or less in wort (or perhaps beer), it should be stored refrigerated. However, if the Brettanomyces will be stored for more than one month in wort (or perhaps beer), it should be stored at room temperature (until more data improves our understanding). Note that at best these storage techniques will decrease viability greatly (80%+) within 3 months, and a starter should be used to try and revive the culture before use ^[184].

Occasional feeding has been shown to keep Brettanomyces alive in beer for brewers who do not have a lab, however many variables may come into play as far as how effective this will be for individual strains and in different environments. Although no research has been done to indicate what the best practices are for feeding Brettanomyces to keep it alive in beer, we recommend trying this method: every 3-6 months swirl the vessel so as to suspend all of the yeast and then decant 70-90% of the beer and suspended yeast slurry, and replace it with a 1.040 starter wort with yeast nutrients. This method will discard a lot of the old yeast cells, while retaining enough living cells for replication ^[185]. Some strains may survive extended periods of aging in beer, however their viability and vitality will be greatly reduced over time. Interestingly, Brettanomyces remains more viable over time if it was co-fermented with S. cerevisiae than if it was fermented without the presence of S. cerevisiae; i.e. 100% Brettanomyces beers or Brettanomyces and Lactobacillus ^[53].

Another method for storing Brettanomyces has reportedly worked for MTF member Justin Amaral. This method involves storing the culture in isotonic sodium chloride. Brettanomyces cultures have been reported by Amaral to survive at least for 6-7 months. This includes other microbes as well (RVA Orchard Brett, ECY Dirty Dozen, Bright Yeast Labs Brett Chateaux, T. delbrueckii, L. plantarum isolated from goodbelly, Omega Lacto blend, Pediococcus damnosus, Bootleg Biology Sour Weapon, and Funk Weapon 2 and 3, and a Brettanomyces isolate from Yeast Bay). For more information on this method, see this Eureka Brewing blog article ^[186].

Tips From Brewers