Difference between revisions of "Brettanomyces"

From Milk The Funk Wiki
Jump to: navigation, search
m (fixed Beta-glucosidases typo)
(added another method for storing Brett)
Line 535: Line 535:
  
 
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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1345924532102424/?comment_id=1345979272096950&reply_comment_id=1346020438759500&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Mark Trent and Richard Preiss on MTF regarding occasional feeding of ''Brettanomyces'' to keep it alive.  07/07/2016.]</ref>.  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%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] or ''Brettanomyces'' and ''Lactobacillus <ref name="Hubbe"></ref>.
 
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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1345924532102424/?comment_id=1345979272096950&reply_comment_id=1346020438759500&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Mark Trent and Richard Preiss on MTF regarding occasional feeding of ''Brettanomyces'' to keep it alive.  07/07/2016.]</ref>.  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%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] or ''Brettanomyces'' and ''Lactobacillus <ref name="Hubbe"></ref>.
 +
 +
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.  For more information on this method, see [https://eurekabrewing.wordpress.com/2012/09/05/yeast-banking-3-isotonic-sodium-chloride/ this Eureka Brewing blog article] <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1631448736883334/ Amaral, Justin.  Milk The Funk Facebook group.  03/30/2017.]</ref>.
  
 
===Tips From Brewers===
 
===Tips From Brewers===

Revision as of 13:17, 30 March 2017

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, in some styles like Saison, lambic, and American sour beers these flavors add a layer of complexity to the beer. Brettanomyces can also form a pellicle. 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 percieve 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 contributes 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]). 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][8][9]. 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.

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 telemorph 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 [10].

Environment and Survival

Brettanomyces has been identified on the skins of fruit (on the skins of cider apples and wine grapes for example) [4][5], as well as vectors (insects) [8]. 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". [11]. 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. Some strains are able to utilize the cellulose of the wood as a carbon source. 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, a pH as low as 2 [12], 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, where as strains isolated from beer did not. This indicated that B. bruxellensis strains living in wine have adapted to the harsher environment of wine [4].

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]) [13]. Some strains of Brettanomyces can metabolize nitrogen sources, such as the amino acids proline and arginine [14]. Some strains of Candida pyralidae, Wickerhamomyces anomalus, Kluyveromyces wickeramii, Torulaspora delbrueckii and Pichia membranifaciens have been found to produce toxin that inhibits Brettanomyces (killer wine strains 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 [15].

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. This was initially dubbed the "negative Pasteur effect" by Custers, and later the "Custers effect" by W. A. Scheffers [16][17].

Carbohydrate Metabolism

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 available of oxygen effects whether or not certain sugars can be fermented by a given strain of Brettanomyces [8][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 [16].

Brettanomyces strains may possess both alpha and beta glucosidases. These enzymes allow Brettanomyces strains to break down a broad range of sugars, including longer chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to Saccharomyces yeasts. [8][18].

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 [16][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 [8]. Some other polysaccharides can be fermented by Brettanomyces, including starch, laminarin, and pectin [14]. 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 [19].

Beta-glucosidases can break down the beta-glycosidic bond in disaccharides (cellulose, cellobiose, and gentiobiose) [20][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 [21]. 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 [14]. 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 [22][23]. For example, some strains are not able to ferment maltose (often B. anomalus strains), which is almost half the sugar content of wort [24]. 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 [14].

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 [25][14].

Just like in other yeast species, temperature has a direct effect on the rate of fermentation for Brettanomyces. The optimal fermentation rate temperature range for Brettanomyces is between 25-32°C (77-90°F). 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. 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) [26]. 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 [27].

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 [23] 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 potentially 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) [28]. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic [29]. It is speculated that flavor compounds from hops can also be released from glycosides [30], however at least one study has shown no significant difference in a blind taste test between hopped beer exposed to beta-glucosidase enzyme and hopped beer that was not exposed to the enzyme [31].

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 [32]. 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. 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 fatty acids, nucleotides, polysaccharides, polypeptides, and other proteins [33]. 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 [34]. 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 it's 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 [35][16]. 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, presence of oxygen, and other nutrients, play a large role in 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].

Ester Production

Brettanomyces is capable of synthesizing several ethyl esters from ethanol and fatty acids. Among the most prolific of these are ethyl acetate, ethyl lactate and phenethyl acetate, along with the hydrolysis (breakdown) of isoamyl acetate. During non-mixed fermentations where lactic acid is minimal to none, insignificant amounts of ethyl lactate ester is 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 varies widely based on species and strain [36]. 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 [37].

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 less 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 [38]. 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 sensory character of the beer [39]. 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) [40]. 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 is 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, some time 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 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 [38]. 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 [39].

See also:

Ester Precursors Flavor/Odor Threshold Molecular Formula Notes
Amyl octanoate (spicy, clove, chemical, plastic) [6] Amyl alcohol and caprylic acid C13H26O2 [41] Also known as pentyl octanoate, it is a flavoring agent [42].
Ethyl acetate (fruity, pineapple, solventy) Acetic Acid and ethanol 33ppm (odor), 100ppm (flavor) C4H8O2 [43] High flavor threshold; pineapple or pear-like in low amounts and nail polish in high amounts. Increases production with higher temperatures and oxygen. Also produced by Saccharomyces species [40].
Ethyl butyrate (pineapple, mango, tropical fruit [44], juicy fruit gum [45]) Butyric Acid and ethanol 0.4ppm (flavor) [46] C6H12O2 [47] Low levels of production by some species of Brettanomyces; production decreases with higher acidity [48]. Also known as ethyl butanoate [47].
Ethyl caproate (sweet, fruity, pineapple, banana, apple or aniseed) Caproic acid and ethanol [49] 0.2ppm (flavor) [50] C8H16O2 [51] Also known as Ethyl hexanoate, Ethyl butyl acetate, and butylacetate [52]. Can also be produced by Saccharomyces species [40].
Ethyl caprylate (Sweet, waxy, fruity and pineapple with creamy, fatty, mushroom and cognac notes [53]) Caprylic acid (contained in buckwheat; produced by yeast autolysis) and ethanol [54] 15ppb (flavor) [55] C10H20O2 [56] Also known as ethyl octanoate [56][57].
Ethyl Decanoate/Ethyl Caprate (brandy, fruity, oily, grape) Decanoic acid (Capric Acid) and ethanol [58] 0.5ppm (flavor in water) [59] C12H24O2 [58] Also known as Ethyl caprate, Ethyl caprinate, and Capric acid ethyl ester [60]. Can also be produced by Saccharomyces species [40]
Ethyl hexanoate [6] (Apple or aniseed [61]) Hexanoic acid and ethanol [62]. 0.2ppm (flavor in beer) [61] C8H16O2 Also produced by both ale and lager yeast; it is a key flavor note in many beers [61]. High amounts Produced by two strains out of 9 B. bruxellensis in one study [6].
Ethyl isobutyrate [6] (Pungent, etherial and fruity with a rum-and egg nog-like nuance [63]) Isobutyric acid and ethanol [64] 0.1 ppb (odor in water) [65] C6H12O2 Also known as Ethyl 2-methylpropanoate, it is found in many alcoholic beverages as well as fruits such as apple, banana, orange, wine grapes, strawberry, and nectarine, and is used as a flavoring agent [66]. Produced in significant amounts by 3 out of 9 strains of B. bruxellensis tested in one study [6].
Ethyl isovalerate (fruity, sweet, berry-like with a ripe, pulpy fruit nuance, artificial grape [67]) [68][69][6] Isovaleric Acid and ethanol 30ppm (flavor) [67] C7H14O2 (same as ethyl valerate) [67] Also found in pineapple, orange juice and peel oil, bilberry, blueberry, strawberry, Swiss cheese, other cheeses, cognac, rum, whiskey, sherry, grape wines, cocoa, passion fruit, mango, and mussels [67]. Also known as Ethyl 3-methylbutanoate [68]. Not identified as a major product of B. bruxellensis, but is produced in large quantities by some strains [6].
Ethyl lactate (fruity, creamy, rum [70][71]) Lactic Acid and ethanol 0.2 ppm-1.66 ppm (odor) [72] C5H10O3 [73] Increases production with higher amounts of Lactic Acid [74]
Ethyl valerate (Sweet, fruity, acidic, pineapple, apple, green, berry, tropical, bubblegum [6][75]) [68][69] Valeric Acid (pentanoic acid) and ethanol 1500-5000 ppm (odor) [76] C7H14O2 [75] Valeric acid quantities found in beer are minimal (0-1 ppm) and below odor threshold [77][78], and is probably also the case for Ethyl valerate. Ethyl valerate is also known as ethyl pentanoate [75]. Also found in apples, bananas, guava, stawberry, cheeses, rum, whiskey, cider, sherry, grape wines, cocoa, coffee, honey, and passion fruit [76]. Not identified as a major product of B. bruxellensis, but is produced in large quantities by some strains [6].
Ethyl-2-methyl butyrate (minty, menthol, citrus, green apple) [6] Ethanol, methanol, and butyric acid C7H14O2 Also known as ethyl 2-methylbutanoate [79]. Found in bilberry, and in many other fruits, e.g.raw and cooked apple, apricot, orange, grapefruit. Used as a fruit flavor additive [80]. Identified as a major product of B. bruxellensis [6].
Isoamyl acetate (banana) Acetic Acid and Isoamyl alcohol 1.1ppm (flavor) [81] C7H14O2 [82] Produced by certain Saccharomyces strains but concentrations are generally reduced by Brettanomyces. Brettanomyces produces only very small amounts itself [38]
Octyl butyrate (spicy, eucalyptus, floral, plastic) [6] Octanol and butyric acid C12H24O2 [83]
Pentyl formate (artificial fruit, candy, chemical) [6] Pentanol and formic acid [84][85] C6H12O2 Also known as amyl formate [86].
Phenethyl acetate (sweet, honey, rose flower like) Acetyl-CoA and 2-phenylethanol [87][88] 3-5ppm (odor), 5-10ppm (flavor) [89] C10H12O2 Produced in very small amounts in Lambic [38][90]. Can also be produced by Saccharomyces species [40]
Phenethyl formate (artificial floral, perfume, wild flower, solvent) [6] 2-phenylethanol and formic acid C9H10O2

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, 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 [91], Brettanomyces is also able to reduce these vinyl derivatives to ethyl derivatives. 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. Some data supports that pitching rate does not have an effect on how much phenol content is produced by Brettanomyces[39]. This contradicts the somewhat popular belief that under-pitching Brettanomyces produces more "funky" flavors.

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 [92][4]. Phenols have been shown to have positive effects on decreasing protein glycation, a complication associated with type 2 diabetes [93].

See also:

Phenol Phenol Type Precursors Flavor/Odor Threshold Molecular Structure Notes
4-Vinylphenol [94] [95] (Barnyard, Medicinal, Band-aid, Plastic) Vinyl phenol p-Coumaric Acid 0.2 ppm (flavor; in beer) [96] C8H8O [97] Production level is different across species/strains of Brettanomyces [98].
4-Vinylguaiacol [94][95] (Clove) Vinyl phenol Ferulic Acid 0.3 ppm (flavor; in beer) [99] Also known as 2-methoxy-4-vinyl phenol [100] C9H10O2 [100]. Produced by some strains of S. cerevisiae [101]. Some Brettanomyces species/strains may also be able to produce this compound at varying levels [68][102][98].
4-Vinylcatechol [94][95] (Plastic, Bitter, Smokey) Vinyl phenol Caffeic Acid C8H8O2 [103] Production level is difference across species/strains of Brettanomyces [98].
4-Ethylphenol [94][95] (Barnyard, Spicy, Smoky, Medicinal, Band-Aid [104]) Ethyl phenol 4-vinylphenol 0.3 ppm (odor; in beer) [105] C8H10O [106] Also known as 1-Ethyl-4-hydroxybenzene and P-Ethylphenol [106]. Identified as a major product of B. bruxellensis [6].
4-Ethylguaiacol [94][95] (Smokey, Spicy, Clove [107]) Ethyl phenol 4-vinylguaiacol 0.13 ppm (odor; in beer) [105] C9H12O2 [108] Also known as 4-Ethyl-2-methoxyphenol [108]. Identified as a major product of B. bruxellensis [6].
4-Ethylcatechol [94][95] (Band‐aide, Medicinal, Barnyard) Ethyl phenol 4-Vinylcatechol C8H10O2 [109] Also known as 4-ethylbenzene-1,2-diol [109].

Another way to read the table above is to list the order in which the precursors are converted into phenol metabolites:

p-Coumaric Acid (found in malt and other ingredients) converts to 4-Vinylphenol by POF+ strains of Saccharomyces and Brettanomyces, which converts to 4-Ethylphenol by Brettanomyces.
Ferulic Acid (found in malt and other ingredients) converts to 4-Vinylguaiacol by POF+ strains of Saccharomyces and Brettanomyces, which converts to 4-Ethylguaiacol by Brettanomyces.
Caffeic Acid (found in malt and other ingredients) converts to 4-Vinylcatechol by POF+ strains of Saccharomyces and Brettanomyces, which converts to 4-Ethylcatechol by Brettanomyces.

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) [40]. 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) [48]. 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-courmaric acid inhibited 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 [91].

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 [91]. 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 [48].
Butyric Acid (Vomit, bile) Associated with supplements of phenylalanine or tyrosine [6] Fatty acid.
Capric acid (Barnyard animal odor/taste) [48][6] Fatty acid. Also found in milk, coconut oil, and seed oils [110]. Also referred to as Decanoic acid [111].
Caproic acid (Fatty, cheesy, waxy, barnyardy) [48][6] Fatty acid. Also known as hexanoic acid [112].
Caprylic acid (Rancid-like smell and taste [48][6] Fatty acid. Also found in milk. Gives a waxy/oily mouthfeel. Flavor is more intense at low pH levels. Also called octanoic acid.[113]
Heptanoic acid (sweaty, solvent, rotten, barnyard) Associated with phenylalanine or tyrosine [48][6] Fatty acid. Also known as enanthic acid [114].
Isovaleric Acid (Feety, parmesian) [115][116] 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) [48] Fatty acid. Also known as dodecanoic acid and dodecylic acid [117].
Nonanoic acid (Rancid odor) [48] Fatty acid. Also known as pelargonic acid [118].
Undecanoic acid (coconut, balsam, oily, vanilla) [48] Associated with tyrosine [6]. Fatty acid. Also known as undecylic acid [119].

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 [120].
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 [121]. 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 [122].
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 [123].
Phenethyl alcohol [6] (floral, dried rose [124]) 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 [125]) Aldehyde
Tetrahydropyridine (Cheerios®, mousy, urine, cracker biscuit, corn chips) Ketone [126] 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 [127].
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 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%. [128]

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 [129].

GigaYeast

Common Name Species Name Synonym (Strain) Name Lab/Package Flavor/Aroma Source Note
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 Source Note
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 TYB 184 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 [130].
Bruxellensis Dekkera bruxellensis B. bruxellensis TYB 207 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 [131].

White Labs

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. [132] Not the same as WY's Brux. Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL [133].
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 [132]. 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 [134] [135]. 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 [133].
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" [132]. Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL [133].
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 [132]. Different from WY's "lambicus". Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL [133].

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 [136]. Cell count: 7.5 x 108 cells/mL [137].
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 [137].
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 [137].
Lambicus Dekkera bruxellensis B. lambicus Wyeast 5526 Pie-cherry Different from WL's "lambicus". Cell count: 7.5 x 108 cells/mL [137].

Smaller Labs

Mfg Package Taxonomy 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 [138].
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 [139]. 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 [140].
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 [139]. 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 [140].
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 [139]. 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 [140].
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 [139]. 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 [140].

Using Brett

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, Funky Mixed Fermentations, 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 [40][141]. 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 [40][142].

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 [26].

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 [143], 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 [144]. 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 [145].

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 [146][147][148]. 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 [149]. 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 and 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 [150].

Maintaining a temperature of 77°-86°F/25°-30°C results in faster growth than lower temperatures and is recommended [151]. Brettanomyces cell growth typically takes about 7-8 days to reach it's maximum growth [152], however some strains may grow at faster rates and finish in 3-4 days [153]. When the starter turns a rich creamy color, it should be done within a few hours after this visual indication occurs [151]. 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 [152][154]. Since yeast calculators are based off of Sacch 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 has 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) [154].

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 continued 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 [155]. Cells grown in MYPG also are better adapted to grow in wort [156]. 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 [157].

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 [40][158].

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 [143][159].

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 [160]. The next best option for long term storage of Brettanomyces is freezing with 10% glycerol. Another 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 [161][162].

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 [163].

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 [164].

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 [165]. 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 [40].

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. For more information on this method, see this Eureka Brewing blog article [166].

Tips From Brewers

The Yeast Bay

  • To make a starter for the Lochristi blend, run it semi-aerobic for 4-6 days in the 70's and then let it settle at room temp and decant what you can if the starter is large [167].

See Also

Additional Articles on MTF Wiki

External Resources

References

  1. Wikipedia. Brettanomyces. Retrieved 2/24/2015.
  2. "When was Brettanomyces discovererd?" Ron Pattenson. Shut Up About Barclay Perkins blog. 06/29/2013. retrieved 08/18/2016.
  3. "Improvements in and connected with the Manufacture of English Beers or Malt Liquors and in the Production of Pure Yeast Cultures for use therein." Patent application by Hjelte Claussen for Brettanomyces. A.D. 1903.
  4. 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Brendan D. Smith, Benoit Divol. June 2016.
  5. 5.0 5.1 5.2 5.3 The wine and beer yeast Dekkera bruxellensis. Anna Judith Schifferdecker, Sofia Dashko, Olena P. Ishchuk, and Jure Piškur. 7 July 201.
  6. 6.00 6.01 6.02 6.03 6.04 6.05 6.06 6.07 6.08 6.09 6.10 6.11 6.12 6.13 6.14 6.15 6.16 6.17 6.18 6.19 6.20 6.21 6.22 6.23 6.24 6.25 6.26 6.27 6.28 6.29 6.30 6.31 6.32 6.33 6.34 6.35 6.36 6.37 6.38 6.39 6.40 Brettanomyces bruxellensis Aroma-Active Compounds Determined by SPME GC-MS Olfactory Analysis. C.M. Lucy Joseph, Elizabeth A. Albino, Susan E. Ebeler, Linda F. Bisson. 2015.
  7. Dekkera, Brettanomyces and Eeniella: Electrophoretic comparison of enzymes and DNA–DNA homology. Maudy Th. Smith, M. Yamazaki, G. A. Poot. 1990.
  8. 8.0 8.1 8.2 8.3 8.4 Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.
  9. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Sam Crauwels, Bo Zhu, Jan Steensels, Pieter Busschaert, Gorik De Samblanx, Kathleen Marchald, Kris A. Willems, Kevin J. Verstrepen and Bart Lieven. 2014.
  10. Brettanomyces acidodurans sp. nov., a new acetic acid producing yeast species from olive oil. Péter G, Dlauchy D, Tóbiás A, Fülöp L, Podgoršek M, Čadež N. 2017.
  11. Screening of yeast mycoflora in winery air samples and their risk of wine contamination. E. Ocón, P. Garijo, S. Sanz, C. Olarte, R. López, P. Santamaría, A.R. Gutiérrez. Food Control Volume 34, Issue 2, December 2013, Pages 261–267.
  12. Wines and Vines. New Research on Role of Yeast in Winemaking; report on a presentation by David Mills and Lucy Joseph from UC Davis. 11/14/2014. Retrieved 08/16/2015.
  13. Removal of Brettanomyces Bruxellensis from Red Wine Using Membrane Filtration. Umiker, Descenzo, Lee, and Edwards. 04/24/2012.
  14. 14.0 14.1 14.2 14.3 14.4 Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains. S. Crauwels, A. Van Assche, R. de Jonge, A. R. Borneman, C. Verreth, P. Troels, G. De Samblanx, K. Marchal, Y. Van de Peer, K. A. Willems, K. J. Verstrepen, C. D. Curtin, B. Lievens. 2015
  15. The influence of thiamine and riboflavin on various spoilage microorganisms commonly found in beer. Barry Hucker, Melinda Christophersen, Frank Vriesekoop. 2017.
  16. 16.0 16.1 16.2 16.3 Yakobson, Chad. The Brettanomyces Project. Introduction. Retrieved 8/11/2015.
  17. On the inhibition of alcoholic fermentation in Brettanomyces yeasts under anaerobic conditions. W. A. Scheffers. 1961.
  18. Insight into the Dekkera anomala YV396 genome. Samuel Aeschlimann. Self published on Eureka Brewing Blog. Spet 2015.
  19. Localization and Characterization of α-Glucosidase Activity in Brettanomyces lambicus. H. M. C. Shantha Kumara, S. De Cort and H. Verachtert. 1993.
  20. "Disaccharides." UC Davis Chemwiki. Retrieved 05/15/2016.
  21. Daenen et al., 2008. Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. FEMS Yeast Res. 8, 1103-1114.
  22. Lance Shaner experiment comparing the growth of various Brettanomyces spp on different growth mediums. 04/07/2016.
  23. 23.0 23.1 National Collection of Yeast Cultures. Search for Brettanomyces. Retrieved 04/07/2016.
  24. "Sugar composition of wort". Eureka Brewing Blog. Jan 13, 2015. Retrieved 04/07/2016.
  25. Determination of sugar metabolism profiles of non-traditional yeasts in the Saccharomyces and Brettanomyces families. J. D. Cook, W. A. DEUTSCHMAN. ASBC Proceeding. 2015.
  26. 26.0 26.1 Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects. Brandam C, Castro-Martínez C, Délia ML, Ramón-Portugal F, Strehaiano P. 2008.
  27. MTF conversation with Richard Preiss of Escarpment Labs. 01/20/2017.
  28. "Glycoside." Wikipedia. Retrieved 06/27/2016.
  29. Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. Luk Daenen, Femke Sterckx, Freddy R. Delvaux, Hubert Verachtert & Guy Derdelinckx. 2007.
  30. Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.
  31. Characterization of the recombinant Brettanomyces anomalus β-glucosidase and its potential for bioflavoring. Yannick Vervoort, Beatriz Herrera-Malaver, Stijn Mertens, Victor Guadalupe Medina, Jorge Duitama, Lotte Michiels, Guy Derdelinckx, Karin Voordeckers, and Kevin J. Verstrepen. 2016.
  32. Principles of Brewing Science, A Study of Serious Brewing Issues. George Fix. Brewers Publications. 1999. Pg 40.
  33. Private correspondence with Richard Preiss by Dan Pixley. 08/23/2016.]
  34. Wikipedia. Secondary Metabolite. Retrieved 6/2/2015.
  35. Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts. Yi-Cheng Guo, Lin Zhang, Shao-Xing Dai, Wen-Xing Li, Jun-Juan Zheng, Gong-Hua Li, Jing-Fei Huang. 2016.
  36. Yakobson, Chad. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Production of Secondary Metabolites. 2011.
  37. "Thoughts on Spitaels and Van Kerrebroeck et al, 2015." Dave Janssen. Hors Catégorie Blog. 02/20/2016. Retrieved 03/15/2016.
  38. 38.0 38.1 38.2 38.3 Spaepen and Verachtert, 1982. Esterase Activity in the Genus Brettanomyces
  39. 39.0 39.1 39.2 Brettanomyces secondary fermentation experiment. Milk The Funk Wiki. Lance Shaner and Richard Preiss. Retrieved 04/21/2016.
  40. 40.0 40.1 40.2 40.3 40.4 40.5 40.6 40.7 40.8 40.9 Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016. Cite error: Invalid <ref> tag; name "Hubbe" defined multiple times with different content
  41. "Amyl octanoate". The Good Scents Company. Retrieved 02/24/2017.
  42. "Amyl octanoate". PubChem website. Retrieved 02/24/2017.
  43. PubChem. Ethyl Acetate. Retrieved 08/15/2015.
  44. Ethyl Butyrate Beer Flavour Standard. FlavorActIV. Retrieved 6/20/2015.
  45. Private corrospondance with Richard Preiss by Dan pixley. 12/1/2016]
  46. Flavoractiv. Ethyl butyrate. Retrieved 1/18/2015.
  47. 47.0 47.1 PubChem. Ethyl Butyrate. Retrieved 08/15/2015.
  48. 48.0 48.1 48.2 48.3 48.4 48.5 48.6 48.7 48.8 48.9 Yakobson, Chad. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Pure Culture Fermentation Discussion. 2011.
  49. Encyclopedia of Food Microbiology. Batt, Carl A. Academic Press. Sep 28, 1999. Pg 320.
  50. Aroxa. ethyl hexanoate. Retrieved 1/18/2015.
  51. PubChem. Ethyl Caproate. Retrieved 08/15/2015.
  52. Chemspider. Ethylhexanoat. Retrieved 1/18/2015.
  53. Ethyl Octanoate. The Good Scents Company. Retrieved 5/28/2015.
  54. Chop & Brew - Episode 37: Influence of Mashing on Sour Beer Production by Michael Tonsmeire. NHC 2014 Presentation. At 26 minutes. Retrieved 5/28/2015.
  55. Esters Detection Tresholds & Molecular Structures. Leffingwell & Associates. Retrieved 5/28/2015.
  56. 56.0 56.1 PubChem. Ethyl Caprylate. Retrieved 08/15/2015.
  57. "Ethyl caprilate". PubChem website. Retrieved 01/30/2017.
  58. 58.0 58.1 Wikipedia. Ethyl Decanoate. Retrieved 1/18/2015.
  59. Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Ralf Günter Berger. Springer Science & Business Media, Mar 6, 2007. Pg 222.
  60. Spedding, Gary. Flavor notes for Michigan Craft Guild Conference. 2014.
  61. 61.0 61.1 61.2 "Ethyl hexanoate". Aroxa Website. Retrieved 01/31/2017.
  62. "Ethyl hexanoate". Wikipedia. Retrieved 01/31/2017.
  63. "Ethyl isobutyrate". The Good scents Company. Retrieved 01/31/2017.
  64. "2-Methylpropanoate". The Good Scents Company Website. Retrieved 01/31/2017.
  65. "Odor & Flavor Detection Thresholds in Water (In Parts per Billion)". Leffingwell & Associates Website. Retrieved 01/31/2017.
  66. "Ethyl Isobutyrate". PubChem Website. Retrieved 01/31/2017.
  67. 67.0 67.1 67.2 67.3 Fenaroli's Handbook of Flavor Ingredients, Fifth Edition. George A. Burdock. CRC Press, Dec 3, 2004. Pg 587.
  68. 68.0 68.1 68.2 68.3 Supplemental Data for: Joseph, C.M.L., E.A. Albino, S.E. Ebeler, and L.F. Bisson. Brettanomyces bruxellensis aroma-active compounds determined by SPME GC-MS olfactory analysis. 2015.
  69. 69.0 69.1 Impact of Brettanomyces on Wine. Presentation by Lucy Joseph of UC Davis. Retrieved 08/15/2015.
  70. Best Aroma website. Ethyl Lactate. Retrieved 08/15/2015.
  71. Dictionary of Flavors. Dolf De Rovira. John Wiley & Sons, Feb 28, 2008. Pg 384.
  72. Haz-Map, Ethyl Lactate odor threshold.
  73. PubChem. Ethyl Lactate. Retrieved 08/15/2015.
  74. Yakobson, Chad. The Brettanomyces Project. Impact of the Initial Concentration of Lactic Acid on Pure Culture Fermentation. Retrieved 6/16/2015.
  75. 75.0 75.1 75.2 The Good Scents Company. Ethyl Valerate article. Retrieved 08/15/2015. Cite error: Invalid <ref> tag; name "goodscents_ethylvalerate" defined multiple times with different content
  76. 76.0 76.1 Fenaroli's Handbook of Flavor Ingredients, Fifth Edition. George A. Burdock. CRC Press, Dec 3, 2004. Pg 638.
  77. Organoleptic Threshold Values of Some Organic Acids in Beer. Sigmund Engan. 1973.
  78. Aroma of Beer, Wine and Distilled Alcoholic Beverages. L. Nykänen, H. Suomalainen. Springer Science & Business Media, May 31, 1983.
  79. "Ethyl 2-methyl butyrate". The Good Scents Company website. Retrieved 02/24/2017.
  80. "Ethyl 2-methylbutyrate". PubChem website. retrieved 02/24/2017.
  81. Aroxa. Isaoamyl acetate. Retrieved 1/18/2015.
  82. PubChem. Isoamyl Acetate. Retrieved 08/25/2015.
  83. "Octyl butyrate". The Good Scents Company website. Retrieved 02/24/2017.
  84. "1-pentanol". Wikiepedia article. Retrieved 02/24/2017.
  85. "Formate". Wikipedia article. Retrieved 02/24/2017.
  86. "Amyl format". Good Scents Company website. Retrieved 02/24/2017.
  87. PubChem. Phenethyl Acetate. Retrieved 08/15/2015.
  88. YMDB. Phenethyl acetate.
  89. Burdock, George A. Fenaroli's Handbook of Flavor Ingredients, Fifth Edition. CRC Press. 2005. pg 1521.
  90. Characterization of aroma and flavor compounds present in lambic (gueuze) beer. Katherine A Thompson Witrick. 2012.
  91. 91.0 91.1 91.2 Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast. Michael Lentz and Chad Harris. 2015.
  92. Olfaction: Smells Like Fly Food. Geraldine A. Wright. 2015.
  93. High Phenolic Beer Inhibits Protein Glycation In Vitro. Susan M. Elrod, Phillip Greenspan, Erik H. Hofmeister. 2017.
  94. 94.0 94.1 94.2 94.3 94.4 94.5 Doss, Greg. Brettanomyces: Flavors and performance of single and multiple strain fermentations with respect to time. Presentation at 2008 NHC. pg 12.
  95. 95.0 95.1 95.2 95.3 95.4 95.5 Yakobson, Chad. Brettanomyces in Brewing the horse the goat and the barnyard. 1/14/2011
  96. Determination of 4-vinylgaiacol and 4-vinylphenol in top-fermented wheat beers by isocratic high performance liquid chromatography with ultraviolet detector. Mingguang Zhu; Yunqian Cui. Dec 2013.
  97. The Good Scents Company. 4-Vinylphenol. Retrieved 08/18/2015.
  98. 98.0 98.1 98.2 Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production. A. Oelofse, A. Lonvaud-Funel, M. du Toit. 2009.
  99. Aroxa Website. 4-Vinylguaiacol. Retrieved 08/19/2015.
  100. 100.0 100.1 The Good Scents Company. 2-methoxy-4-vinyl phenol. Retrieved 08/18/2015.
  101. Ferulic Acid Release and 4-Vinylguaiacol Formation during Brewing and Fermentation:  Indications for Feruloyl Esterase Activity in Saccharomyces cerevisiae. Stefan Coghe, Koen Benoot, Filip Delvaux, Bart Vanderhaegen, and Freddy R. Delvaux. 2004.
  102. The biotransformation of simple phenolic compounds by Brettanomyces anomalus. Duncan A.N. Edlin1, Arjan Narbad, J. Richard Dickinson1 andDavid Lloyd. 2006.
  103. PubChem. 3-Vinylcatechol. Retrieved 08/18/2015.
  104. Aroxa Website. 4-Ethyl Phenol. Retrieved 08/19/2015.
  105. 105.0 105.1 Volatile Compounds in Foods and Beverages. Henk Maarse. CRC Press, Mar 29, 1991. Pg 514, 515.
  106. 106.0 106.1 PubChem Website. 4-Ethylphenol. Retrieved 08/19/2015.
  107. Aroxa Website. 4-Ethyl guaiacol. Retrieved 08/19/2015.
  108. 108.0 108.1 PubChem Website. 4-Ethyl-2-methoxyphenol. Retrieved 08/19/2015.
  109. 109.0 109.1 PubChem Website. 4-Ethylcatechol. Retrieved 08/19/2015.
  110. "Decanoic acid". Wikipedia.
  111. "Decanoic acid". Wikipedia. Retrieved 02/01/2017.
  112. Hexanoic acid. The Good Scents Company website. Retrieved 03/01/2017.
  113. FlavorActV. Caprylic Acid. Retrieved 2/10/2015.
  114. Heptanoic acid. PubChem website. Retrieved 03/01/2017.
  115. Botha, Janita J. Sensory, chemical and consumer analysis of Brettanomyces spoilage in South African wines. March 2010. Pg 2, 13, 17, 18
  116. Oelofse, Adriaan. Investigating the role of Brettanomyces and Dekkera during winemaking. December 2008.
  117. Lauric acid. PubChem website. Retrieved 03/01/2017.
  118. Nonanoic acid. PubChem website. Retrieved 03/01/2017.
  119. Undecanoic Acid. PubChem website. Retrieved 03/01/2017.
  120. "1-decanol". PubChem Website. Retrieved 01/30/2017.
  121. "3-Methyl-1-butanol". PubChem Website. Retrieved 01/30/2017.
  122. "Nerolidol". PubChem Website. Retrieved 01/30/2017.
  123. "1-octanol". PubChem Website. Retrieved 01/30/2017.
  124. "Phenethyl alcohol". The Good Scents Company Website. Retrieved 01/30/2017.
  125. "Phenyl acetaldehyde". The Good Scents Company Website. Retrieved 01/30/2017.
  126. Humbard, Matt. Milk The Funk Discussion. 3/10/2015.
  127. Conversation with Mark Horsley and Ed Coffey on MTF. 02/11/2016.
  128. Imperial Organic Yeast website. Retrieved 09/14/2016.
  129. "INIS-950 BRETTANOMYCES COPENHAGEN". Inland Island website. retrieved 02/24/2017.
  130. BRETTANOMYCES SINGLE STRAIN SERIES - ISOLATE TYB 184. The Yeast Bay website. Retrieved 07/27/2016.
  131. [http://www.theyeastbay.com/wild-yeast-and-bacteria-products/brettanomyces-single-strain-series-isolate-tyb-207 BRETTANOMYCES SINGLE STRAIN SERIES - ISOLATE TYB 207. The Yeast Bay website. Retrieved 11/16/2016.
  132. 132.0 132.1 132.2 132.3 Descriptors put together by Dan Pixley and Travis Trembly during a spit batch tasting. 12/28/2016.
  133. 133.0 133.1 133.2 133.3 Homebrewing Reddit post. 02/26/2016.
  134. Ryan Steagall's conversation with White Labs on Facebook. April 10, 2015.
  135. White Labs 644 Explanation. 04/09/2015. Retrieved 5/2/2015.
  136. "All About Brett." Michael Tonsmeire. Brew Your Own Magazine. October 2016.
  137. 137.0 137.1 137.2 137.3 Wyeast Specifications 2015 Retail Products. 2015.
  138. Conversation with Lance Shaner on MTF regarding Omega cell counts. 10/09/2015.
  139. 139.0 139.1 139.2 139.3 Conversation with Nick Impellitteri regarding taxonomy of the TYB Brett blends. 11/13/2015.
  140. 140.0 140.1 140.2 140.3 Conversation on MTF with Nick Nick Impellitteri from The Yeast Bay. 01/16/2017.
  141. MTF post by Brian Martyniak on Brettanomyces growth in competition with Saccharomyces under aerobic and anaerobic conditions. 07/29/2016.
  142. MTF Brettanomyces Propagation Experiment
  143. 143.0 143.1 Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Methods. Retrieved 2/18/2015.
  144. Conversation with Mark Trent, Richard Preiss, and Roy Ventullo on MTF regarding creating a semi-aerobic starter without an orbital shaker. 11/06/2015
  145. Conversation with Nick Impellitteri on MTF in regards to semi-aerobic starters. 2/16/2015.
  146. Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. Aguilar Uscanga, Délia1, and Strehaiano. 2003.
  147. Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking. Maurizio Ciani and Luisa Ferraro. April 1999.
  148. Acetic acid production by Dekkera/Brettanomyces yeasts. S.N. Feer. April 2002.
  149. Conversation with Richard Preiss of Escarpment Yeast Labs on MTF. 6/26/2015.
  150. Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.
  151. 151.0 151.1 Conversation with Mark Trent and Richard Preiss on optimal temperatures for Brett starters on MTF. 06/21/2016.
  152. 152.0 152.1 Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Results. Retrieved 2/17/2015
  153. Brettanomyces_Propagation_Experiment
  154. 154.0 154.1 Conversation with Mark Trent and Lance Shaner on MTF regarding Brett pitching rates. 07-21-2015.
  155. Yakobson, Chad. The Brettanomyces Project. MYPG Compared to Wort as a Growth Substrate. Retrieved 2/18/2015.
  156. Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Discussion. Paragraph 5. Retrieved 2/18/2015.
  157. Conversation with Mark Trent, Lance Shaner, and Richard Preiss on MTF. 09/18/2015.
  158. Discussion with Richard Preiss regarding counting Brettanomyces with methylene blue versus trypan blue staining. 09/15/2016.
  159. Discussion with Mark Trent on Milk The Funk. 4/2/2015.
  160. Cryopreservation. Wikipedia. Retrieved 07/07/2016.
  161. Conversation with Matt Humbard, Ritchie Preiss, and Jeff Melo on MTF. 6/4/2015.
  162. Conversation with Nick Impellitteri on MTF regarding storing Brett on agar plates. 7/24/2015.
  163. Yakobson, Chad. Presentation at 2012 Music City Brew Off. At 43:00.
  164. Brettanomyces_Storage_Survival_Experiment
  165. Conversation with Mark Trent and Richard Preiss on MTF regarding occasional feeding of Brettanomyces to keep it alive. 07/07/2016.
  166. Amaral, Justin. Milk The Funk Facebook group. 03/30/2017.
  167. Nick Impellitteri of The Yeast Bay on a MTF thread. Feb 17, 2015.