Brettanomyces

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, although this form is extremely rare or perhaps even non-existent . Known for its barnyard, fecal, horsey, metallic or Band-Aid flavors, Brettanomyces continues to be unwelcome in many breweries and most wineries . However, Brettanomyces also produces high levels of fruity esters that are desirable in some styles like saison, lambic, and American sour beers. Brettanomyces can also form a pellicle during fermentation. See Lactobacillus, Pediococcus, Saccharomyces, Mixed Cultures, Kveik, and Nonconventional Yeasts and Bacteria charts for other commercially available cultures.

Introduction of History, Characteristics, and Taxonomy

Closely related to Saccharomyces, Brettanomyces diverged from its cousin yeast more than 200 million years ago. Both genera evolved independently to ferment sugar and produce ethanol . Although first isolated in 1889 and again in 1899 by scientists at Guinness, the discovery of Brettanomyces was first publicly published by the Director of laboratory of the New Carlsberg Brewery, Hjelte Claussen, in 1904 after he cultured it in 1903 from English beers that exhibited a sluggish secondary fermentation . 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 . Brettanomyces, along with dry hop creep, was identified as the source of secondary fermentation during long aged ales, contributing to their lasting high carbonation . Brettanomyces was typically included in 1800's English Stock Ales, Pale Ales, aged porters, and barrel aged IPA's that were shipped to India, and it was considered an important component of both flavor and protecting the beer from contaminants due to the Brettanomyces fermenting the majority of residual sugars (~56 and 59 mins in ) (45 minutes in). Although Claussen saw the character from Brettanomyces as a desirable character in ales and identified its character as a quality of traditional English ales, as pure cultures of Saccharomyces were introduced in English brewing in the early 20th century, Brettanomyces became identified as a contaminate in both wineries and breweries due to some of the phenols, acids, and haze that it sometimes produces. These phenols and acids have generally been described as "barnyard", "burnt plastic", "wet animal", "fecal", and "horse sweat", although some tasters describe these flavors with different terminology because they perceive certain flavor compounds differently while some other tasters simply cannot detect certain flavor compounds at all . 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. Some winemakers and wine tasters have also described wines with certain flavor compounds derived from Brettanomyces as positive characteristics of some wines . It is important to keep in mind that individual tasters on tasting panels describe some flavor compounds as "negative" while others describe them as "positive" (and sometimes a mixed response is given by a taster in regards to a certain flavor compound), and this discrepancy in interpretations of Brettanomyces derived flavor compounds appears to be based on personal preference and experience. In some cases, for example for vinyl phenols, low levels that are not detectable by some people, but detected by others contribute positively to wine, while higher amounts contribute negatively. Thus, a 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 .

See also:

• A collection of early 1900's papers by Claussen and Alfred Chapman; MTF post by Cory Widmayer.
• Yvan de Baets believes that Brettanomyces probably played an important role in historical saison.

Taxonomy

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 ). The species previously known as B. intermedius and B. lambicus have recently been genetically analyzed and reclassified as strains of B. bruxellensis . 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 . All of the other names such as the ones often used by yeast labs (e.g. "claussenii") are derived from old nomenclature that is no longer used scientifically (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 Spain and Israel; however, its presence in olive oil has been described as "rare" because only two strains were found after searching dozens of olive oils. Its closest relation is to B. naardenesis by 73% of its genetic makeup. No teleomorph form was observed. This species is a strong acetic acid producer, and it is very tolerant of acetic acid in its environment. It can consume lactose and cellobiose but does not consume maltose. it is unknown but a possibility that this species contributes to the vinegary taste of spoiled olive oils, although this has generally been attributed to acetic acid bacteria .

A genetic survey of 145 different strains of B. bruxellensis from 29 countries, 5 continents, and 9 different fermentation niches was conducted in 2018 by Avramova et al. They found that these strains formed roughly 6 genetic groups with mostly separate ancestral lineages, and 1 group with a mixed ancestral lineage: 3 wine groups, 1 beer group, 1 kombucha group (most distantly related to the beer group), as well as 1 tequila/ethanol group that has multiple ancestral lineages . These groups are partially determined by the identification of at least two hybridization events that happened during the evolution of B. bruxellensis, similar to the hybridization events that created the Saaz and Frohberg subgroups of S. pastorianus (the parents of these hybridization events in B. bruxellensis, whether from different species or not, has yet to be determined and will require whole genome sequencing of species closely related to B. bruxellensis) . This was expressed mostly in the ploidy level of each group (the number of sets of chromosomes), with 2 of the wine groups, the tequila group, and the beer group containing more sets of chromosome pairs than the other groups (diploid vs triploid; this is thought to encourage adaption and hybridization). Additionally, the triploid wine group was generally more tolerant of SO² than the diploid wine groups .

Colomer et al (2020) surveyed the whole genomes of 64 strains of B. bruxellensis, 14 strains of B. anomalus, 3 strains of B. custersianus, and 3 strains of B. naardenensis. They broke the B. bruxellensis beer group into two clades: a "farmhouse" clade which comprised of strains of B. bruxellensis isolated from commercial craft beer breweries, and a "lambic" clade which comprised of strains isolated from spontaneously fermented Belgian lambic beers. There was also a subdivision of the lambic clade which comprised of strains of B. bruxellensis identified with various natural origins; ethanol plants, barrel-aged beers, and matured wines. This subclade was called "wild/wood" by the researchers. See Figure 2 (family tree) from the study .

Science has also begun to explore targeted gene manipulation of B. bruxellensis via CRISPR, which will eventually lead to a better understanding of the Brettanomyces bruxellensis genome .

Morphology

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

See also:

• Brettanomyces morphology examples from Remi Bonnart.
• "Brett Trois – A riddle, wrapped in a mystery, inside an enigma," Sui Generis Blog; an example of S. cerevisiae appearing like Brettanomyces cells under a microscope.
• Morphology examples on Bootleg Biology's website.

Culturing

See Laboratory Techniques.

Environment and Survival

Up until recently, Brettanomyces has been only occasionally identified on the skins of fruit (on the skins of cider apples and wine grapes for example) , and is thought to disperse via insects such as bees and fruit-flies (called "vectors" in the scientific literature) . More recently, techniques have been invented to more easily isolate Brettanomyces from nature, which might be because of a "VBNC" state . See Wild Brettanomyces for more information. However, the occurrence of Brettanomyces has been more commonly identified in industrial food processing areas (wine, beer, kombucha, soft drinks, dairy products, tea, sourdough, etc.) . For example, B bruxelensis, B. anomala, and B. custersianus have mostly been isolated from wine or beer production, while B. naardenensis has mostly been isolated from soda production . Brettanomyces is not considered to be airborne; however, one study has demonstrated 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". 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 potential 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. Brettanomyces has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and occasionally as deep as 5 to 8 mm, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and one study found that the Brettanomyces was able to penetrate the French oak barrels up to 8 mm, while only penetrate American oak barrels up to 4 mm) of the barrel , with the highest concentration of surviving cells being at the top staves where oxygen is more accessible (although Cartwright et al. found the opposite was true, perhaps due to methodology of sampling or a difference in SO₂ concentrations). Some strains are able to utilize the cellulose of the wood as a carbon source, and occasionally form pseudohyphae within the wood which expands the surface area of the cells allowing them more access to nutrients and allowing them to survive in nutrient deficient environments . Ozone gas has been shown to be an effective way to kill Brettanomyces that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of Brettanomyces on wine grapes. Liquid ozone has been shown to be less effective at eliminating Brettanomyces. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing Brettanomyces within the wood of barrels (see Barrel Sanitation for information on pasteurizing barrels) . 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 .

Unlike most genera of yeast, Brettanomyces has the characteristics of being very tolerant to harsh conditions, including high amounts of alcohol (up to 14.5-15% ABV ), a pH as low as 2 , and environments with low nitrogen and low sugar sources . It has been reported that some strains require a very low concentration of fermentable sugars (less than 300 mg/L) and nitrogen (less than 6 mg/L), which is less than most wines contain . Some strains are able to utilize ethanol, glycerol, acetic acid, and malic acid when no other sugar sources are available . 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 . One study from Dr. Charles Edwards found that a combination of keeping wine under 54°F (12.2°C) and alcohol at or above 14% resulted in a decline of B. bruxellensis populations for up to 100 days for two strains that were tested. The study found that neither of the strains grew well at 14% and stopped growth completely at 16% ABV in wine, but one strain grew better than the other at 15%, demonstrating the genetic diversity of Brettanomyces. The researchers concluded that a combination of high ethanol and cold temperatures as well as sulfur dioxide, chitosan, and filtration could be used to control Brettanomyces in winemaking. Brettanomyces has been found to be able to grow at temperatures as low as 50°F (10°C) and as high as 95°F (35°C); see fermentation temperature for more information .

The genetic diversity of Brettanomyces is particularly wide. For example, one study that analyzed the whole genomes of 53 strains of B. bruxellensis found that the overall genetic diversity between different strains of B. bruxellensis was higher than strains of S. cerevisiae (however, the entire gene set, known as the pangenome, of all the genes among all of the strains of B. bruxellensis is much smaller than the entire gene set of S. cerevisiae) . Some studies have indicated that strains of B. bruxellensis have adapted to specific environments. For example, one study found that strains of B. bruxellensis isolated from wine had 20 genes involved in the metabolism of carbon and nitrogen, whereas strains isolated from beer did not. This indicated that B. bruxellensis strains living in wine have adapted to the harsher environment of wine . Another study found that one out of the two strains tested that were isolated from soda could not ferment maltose, and only strains isolated from wine were able to grow in wine and the beer/soda strains did not. The wine strains were also more resistant to sulfites, which are commonly used in the wine industry to prevent microbial contamination . The whole genome sequencing of one strain of B. naardenensis and lambic strains of B. bruxellensis found that they are missing the genes associated with nitrate utilization, indicating that the assimilation of nitrates is not required to survive in beer, perhaps because of the abundance of nitrogen from other sources found in beer .

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 . Other vitamins such as p-aminobenzoic acid (PABA), folic acid (vitamin B9), nicotinic acid (vitamin B3), pantothenic acid (vitamin B5) are also not required for most strains of Brettanomyces to grow. The presence of alcohol can increase the dependence on vitamins for some strains of Brettanomyces to grow. For example, Myo-inositol (vitamin B8) and thiamine (vitamin B1) were required by two strains of B. bruxellensis when grown in 10% ethanol but not in 0% ethanol. Biotin (vitamin B7) is the one exception, and it was found that the lack of biotin inhibited the growth of some strains of B. bruxellensis. Other studies contradict these previously mentioned findings, showing that thiamine was not required by the strains of B. bruxellensis tested, that pyridoxine was required, and biotin was not required. These discrepancies between scientific studies are probably due to the genetic differences between the strains selected, the growth media chosen by the scientists, and/or the growth conditions .

Sulfur Tolerance

Sulfite and SO₂ inhibits the growth of Brettanomyces, and is often used in the wine industry to prevent the growth of Brettanomyces (although Brettanomyces is usually seen is a contaminant in wine, 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 ) . However, it has been shown that wine strains of B. bruxellensis could survive dosages of up to 1 mg/L of molecular SO₂, and the very high dosage of 2.1 mg/L was needed to kill Brettanomyces in wine . This dosage of molecular SO₂ requires a total amount of SO₂ that is beyond legal limits (350 mg/l ; see this Cornell University blog post that explains the difference between free and molecular SO₂) and has negative effects on wine. One study found that out of 145 strains of B. bruxellensis, 107 of which were wine strains with the rest being from beer, tequila, kombucha, etc., 36% of them were either tolerant (lagged growth, but achieved full growth eventually) or resistant (no lagged growth, and achieved full growth) to 0.6 mg/L of molecular SO₂. 46 of the 52 resistant/tolerant strains were wine strains, thus demonstrating that wine strains of B. bruxellensis are generally more tolerant of SO₂ than strains of B. bruxellensis that are found in other types of beverages. It has been demonstrated that the wine strains have adapted to the conditions of winemakers adding SO₂ to wine . There is also variability between wine strains in their ability to tolerate SO₂ . For example, Brettanomyces strains isolated from sweet wine tend to be more tolerant of sulfur dioxide than strains isolated from dry wine . In addition, it has been proposed that SO₂ and other currently unidentified environmental stress factors can induce a so-called "viable but nonculturable" (VBNC) state in Brettanomyces, which means that Brettanomyces cells in this state cannot grow or be cultured on traditional media but can remain viable and create a low amount of phenol character (see VBNC in Yeast) . Some strains of Candida pyralidae, Wickerhamomyces anomalus, Kluyveromyces wickeramii, Torulaspora delbrueckii and Pichia membranifaciens have been found to produce toxin that inhibits Brettanomyces, and these toxins have been proposed as an alternative to SO₂ as a way to kill Brettanomyces (killer wine strains of Saccharomyces cerevisiae do not kill Brettanomyces; see Killer Wine Yeast for more information). Kaolin silver complex (KAgC) has been found to inhibit Brettanomyces and acetic acid bacteria in wine when used in legal dosages, and has been proposed as a replacement for SO₂ or to minimize the use of SO₂ . Other proposed replacements for SO₂ as a way to inhibit Brettanomyces in wine include high pressure processing and pulsed electric fields .

A study by Cibrario et al. (2019) looked at the genome of B. bruxellensis strains in bottles of wine going back as far as the year 1909 revealed that the SO₂ tolerant triploid strains only started appearing after the year 1990, which corresponds to when the wine industry started using SO₂ in most wine production (although it can also mean that the triploid strains are not as good at surviving in bottles of wine long-term compared to the diploid strains that have been isolated from much older bottles of wine; this will be determined to be the case or not in the future as bottles of wine from the 1990s continue to age). They also identified dozens of examples of wineries throughout France and Italy where the same strain of B. bruxellensis was found in multiple vintages of bottles of wine going back many decades, indicating that individual B. bruxellensis strains become long-term residents in wineries. Some identical strains have been found in different regions and even different continents, indicating that some strains have traveled not just due to traditional vectors such as insects or birds, but also probably due to human transportation such as wine bottle imports/exports or exchanges between industrial processes. This also indicates that while B. bruxellensis becomes rather sedentary and a constant resident in wineries, it can also adapt to the different winemaking conditions in different regions once it's been transported (different grapes, different climates, different fermentation temperatures, etc.), including adapting to improved modern hygienic practices such as higher SO₂ treatment. Overall, the results from this study suggest that Brettanomyces is able to adapt to living alongside certain human industries and has done so for at least a couple of centuries . The genetic differences between the fermentation substrates (beer, wine, etc.) were lower but still significant, and this was explained by the frequent cross-over of equipment such as wine barrels being used for beer fermentation. When comparing the geographic differences, they found geography contributed only 5% of genetic differences, while geography explained more than 50% of genetic differences in non-wine strains, suggesting that beer, kombucha, and tequila strains are more localized genetically than wine strains and that humans probably helped the wine strains travel across the globe. They also found that although one study reported spore-forming versions of B. bruxellensis (referred to as Dekkera bruxellensis), the genetic makeup of the analyzed strains determined their ability to sporulate to be non-existent or rare (only one study that we know of by Walt and Kerken in 1960 has reported sporulation in Brettanomyces only on specific agar types with vitamins added, indicating that sporulation in Brettanomyces is extremely rare) . See also Richard Preiss's discussion of this study on MTF. A study by Bartel et al. (2021) used laboratory adaptive evolution methods to demonstrate that some strains of B. bruxellensis strains did in fact adapt to SO₂ usage in wineries .

See also:

• Calculations for SO₂ additions and associated MTF discussion.
• Viable But Nonculturable Yeast.

Biofilm

First evidence of possible (unconfirmed ) chlamydospore cell structures of B. bruxellensis, found in a biofilm. Photo by Lebleux et al. (2019) .

Brettanomyces has the ability to form a biofilm. Biofilm formation is a survival mechanism induced by stress whereby the cells adhere to non-living surfaces such as plastic and stainless steel . After adhesion to the surface, the cells produce a protective layer of proteins and polysaccharides that help protect the organism from cleaning and sanitizing agents.

Joseph et al. (2007) tested 36 wine strains of B. bruxellensis for biofilm formation over a 10 day period. They found that just under half of the strains formed a biofilm, and about half of those formed considerable and consistent biofilms throughout the tests. Almost all strains tested (95%) adhered to a surface with 0.1% glucose within 6 hours of contact (the same conditions that get Saccharomyces cerevisiae to adhere to a surface; longer contact with surfaces and higher residual sugar could encourage Brettanomyces to adhere more readily to surfaces). A juice-based growth media in the range range of 2 - 4.5 pH was tested for biofilm formation and 3-4 for cell adhesion to a surface, and for most of the strains they formed stronger biofilms and adhered better in the higher pH growth media (4.5 pH being the highest tested). Under a pH of 3.5 significantly dropped biofilm formation and adherence, indicating that something about pH affects the cells ability to attach themselves. The researchers concluded that winemakers should keep wine in the lower end of the pH range (3.5). Six different types of cleaners were tested to see how well they removed the biofilms: keytones + surfactant detergent, quaternary ammonia + surfactant detergent, sodium hydroxide (caustic soda), sodium carbonate (soda ash), sodium hydroxide + surfactant (alkaline detergent), and chlorine (sanitizer, not a detergent). They found that only caustic soda was consistently efficient at removing the biofilm. The chlorine, while it did not remove the biofilm, still killed all of the Brettanomyces cells, and it was presumed that the other cleaners might have killed the Brettanomyces, but that was not tested for. They also tested to see if cells that were adhered to a surface could be cleaned. Again, the caustic soda performed consistently the best, but the ammonia + surfactant cleaner and the quaternary ammonia + surfactant detergent also effectively removed adhered cells. The other cleaners varied in how well they removed adhered cells from a surface .

Dimopoulou et al. (2019) studied Brettanomyces bruxellensis biofilms from each of the genetic branches of B. bruxellensis. They found that for the wine strains biofilms formed more readily when grown in wine must rather than YPD media; however, the beer strains grew biofilms equally well in wine must and YPD media. The biofilms contained a large portion of saturated fatty acids and a smaller portion of monounsatured fatty acids. The amount of exopolysaccharide produced varied widely across the strains tested per cell population, with some wine strains producing little EPS (40 mg/L/OD), beer strains producing moderate amounts, and the other wine group producing a high amount (100 mg/L/OD). Additionally, the different strains displayed a varying degree of negative cell wall charges, with the beer and tequila strains being more negatively charged than wine strains, which could help them adhere to surfaces and form biofilm .

Lebleux et al. (2019) measured biofilm density for 12 strains across 5 of the genetic groups of B. bruxellensis. All of the strains produced a biofilm when in contact with a surface (polystyrene and stainless steel, in the case of this study), and the thickness of the biofilm was proportional to the cell size of each strain. The biofilms contained filamentous cells that started from the base of the biofilm and extended upward, indicating multiple layers. The biofilms also contained exopolysaccharides (EPS), but the makeup of the EPS was not analyzed and this was identified as a goal for further study. The average thickness was only 9.45 µm which is much thinner than other biofilm-forming yeast species (Candida and a biofilm-producing strain of S. cerevisiae ). They found that one or two strains were less dense (contained fewer cells) than the average. A couple of the strains grew a biofilm a little slower than average. Two of the strains in biofilm form were added to wine; each of the biofilms released cells into the wine, although one strain released more cells than the other. Introduction to the wine first led to cell death for some cells due to the harsh environment of the wine, but after several days the B. bruxellensis strains began to re-grow in the wine. It was observed that for one of the strains, the cells appeared larger than normal, round, and had thicker cell walls, possibly forming what is known as chlamydospore cell structures. It was not confirmed in the study whether these cells were actually chlamydospores, and their structure could be due to relatively insignificant reasons . Chlamydospore cell structures are known to help certain species of non-yeast fungi survive harsh environments; however, it has not yet been established that yeast with chlamydospore cell structures helps them survive harsh conditions, and this was also identified in the study as an area for further research .

See also:

• Quality Assurance
• Pellicle

Brettanomyces Metabolism

Dr. Custers memorial. Photo by Jan Beekaa Lemmens.

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) . Perhaps the most differentiating characteristic of Brettanomyces is its preference to ferment glucose in the presence of oxygen to produce ethanol and acetic acid, which is the opposite preference in Saccharomyces where the presence of oxygen inhibits fermentation (dubbed the "Pasteur effect"). Also opposite of most yeasts including Saccharomyces, in a completely anaerobic environment Brettanomyces ceases alcoholic fermentation for about 7-8 hours before adapting to the anaerobic conditions . This was initially dubbed the "negative Pasteur effect" by Custers, and later the "Custers effect" by W. A. Scheffers . A notable exception to this is the species B. naardenensis, which only produces ethanol when oxygen is limited .

Despite the Custers effect in Brettanomyces, this genus is not classified as an "oxidative yeast" but rather as a "fermentative yeast" since oxidative yeasts produce little to no ethanol in the presence of glucose, and only grow as scum on the surface of a liquid rather than within the liquid .

The Custers effect is abolished under anaerobic conditions when nitrate is available. Under conditions where there is no oxygen, as long as nitrates are available, it has been shown that Brettanomyces can produce ethanol just as capably as S. cerevisiae . Brewers who prefer the character of Brettanomyces in their beer can take advantage of this ability by limiting oxygen and providing a food source for Brettanomyces (aka beer or wort). See Nitrogen Metabolism below.

Carbohydrate Metabolism and Fermentation Temperature

Brettanomyces is able to ferment a wide range of sugars. All strains can ferment glucose, and many strains can ferment sucrose, fructose, and maltose, although at a slower rate than glucose. The ability of Brettanomyces to produce invertase enzyme which breaks sucrose down into glucose and fructose has been attributed to horizontal gene transfer from an unknown bacteria at some point in the evolution of Brettanomyces . Some strains can also ferment galactose, mannose, ethanol, acetic acid, malic acid, and glycerol, although historically there are some contradicting studies in science regarding the specifics (more recent studies tend to use better methods), probably due to the genetic diversity of Brettanomyces species, and many previously published studies do not specify whether testing conditions were aerobic or anaerobic even though the availability of oxygen effects whether or not certain sugars can be fermented by a given strain of Brettanomyces . For example, the species B. naardenensis can ferment a wide range of carbon sources, including galactose, maltose, xylose, trehalose, cellobiose, rhamnose, and arabinose . Acetic acid, glycerol, succinic acid, and ethanol are only consumed if oxygen is present . The addition of H+ acceptors such as acetaldehyde, acetone, pyruvic acid, and other carbonyl compounds, stimulates anaerobic fermentation. Small amounts of oxygen also stimulate fermentation . The presence of small amounts of oxygen can allow some strains of Brettanomyces to utilize certain carbon sources. For example, several strains of B. bruxellensis can consume ethanol, glycerol, and acetic acid as food sources only when at least a low amount of oxygen is present (semi-aerobic conditions) and no other sugar is available. Acetic acid and glycerol are used as food sources by some strains only under fully aerobic conditions, but not under semi-aerobic or anaerobic conditions. It has been hypothesized that acetic acid and glycerol are only consumed by Brettanomyces when ethanol and other food sources are no longer available .

Brettanomyces strains may possess both alpha and beta-glucosidases. Beta-glucosidase is intracellular (works on sugars that are passed into the cell through the cell wall), while alpha-glucosidase is both intracellular and extracellular (released into the environment by the cell). These enzymes allow Brettanomyces strains to break down a broad range of sugars, including long-chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to Saccharomyces yeasts. .

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 . 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 . Some other polysaccharides can be fermented by Brettanomyces, including starch, laminarin, and pectin . 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 . A survey of 84 strains from several species of Brettanomyces showed that there is a wide variability in the ability of different strains to ferment maltose, with some strains not being able to ferment it at all and others fermenting it very slowly, suggesting that alpha-glucosidase is not functional or poor in some strains. Additionally, when maltose is present instead of just glucose, the researchers saw an increased lag during the growth phase .

B. bruxellensis and B. nanus can also produce oligo-1,6-glucosidase, which hydrolyze the alpha-1,6 linkages in starch and glycogen to produce oligosaccharides, and then further break down these oligosaccharides to produce sugars with alpha-1,4 linkages (for example, maltose, in the case of starches from malted barley ). These alpha-1,4 linkages (maltose) are then further broken down by the maltose enzyme by strains of Brettanomyces or other present microbes that produce this enzyme . Unlike for some domesticated diastatic S. cerevisiae strains, this ability for Brettanomyces to break down starches has occurred in the wild without domestication .

Beta-glucosidases can break down the beta-glycosidic bond in disaccharides (cellulose, cellobiose, and gentiobiose) , 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 . 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 . 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 . For example, some strains are not able to ferment maltose (often B. anomalus strains), which is almost half the sugar content of wort . 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, in one study 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 (wine barrels). Further studies are needed to see if this is a trend throughout the species . Daenen et al. (2007) found that none of the B. bruxellensis strains isolated from lambic that they tested could utilize cellobiose (see glycosides below). This data point challenges the belief that Brettanomyces lives in wooden barrels because it is able to consume the cellobiose of the wood. A study by Tyrawa et al. from Escarpment Laboratories agreed that wine isolated strains were generally better at fermenting cellobiose than strains isolated from beer at 15°C (59°F), however at 22.5°C (72.5°F) some of the beer strains started to utilize cellobiose, indicating that temperature plays a role in whether Brettanomyces can ferment certain sugars . Colomer et al. (2020) surveyed the whole genome of 64 strains of B. bruxellensis and found that beer strains are more likely to be able to ferment maltose (alpha-glucosidase) but not cellobiose (beta-glucosidase) and wine/wild strains tend to have the opposite tendency, indicating that B. bruxellensis strains have adapted to the environments in which they live over time .

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 .

Just like in other yeast species, the temperature has a direct effect on the rate of fermentation for Brettanomyces. The optimal fermentation rate temperature range for Brettanomyces is between 22-32°C (77-90°F). However, one study by Tyrawa et al. found that several strains of B. bruxellensis fermented at 30°C "smelled terrible" . At 20°C (68°F) fermentation rate is about half as slow. Brettanomyces will still grow at temperatures as low as 15°C (59°F) with about a third of strains being able to grow as low as 10°C (50°F) but growth will be much slower. However, one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth and fermentation temperature range of 20-32°C. The growth rate at 15°C, while still slowly active, varies from strain to strain with some strains growing very poorly. Carbohydrates are consumed much slower, with cellobiose metabolizing ceasing for some strains (although phenol production stayed the same between 15°C and 22.5°C) . At a temperature of 35°C (95°F), fermentation is greatly inhibited due to cell death for most strains of B. bruxellensis, with about a third of strains able to grow as high as 37°C (98.6°F) , and complete elimination in wines at 50°C for 5 minutes (see also Barrel Sanitizing and Pasteurization) . B. naardenensis is less tolerant to extreme temperatures, and it has been demonstrated that this species cannot grow at 30°C or higher . 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) . 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 . For more information on how fermentation temperature affects the flavor compounds of 100% Brettanomyces fermentation, see Impact of Fermentation Temperature.

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	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 .

Glycosides and Beta-Glucosidase Activity

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

Beta-glucosidase also allows the breakdown of cellobiose and cellotriose . This has been believed to be a mechanism in which Brettanomyces can survive in barrels; however, most strains of Brettanomyces found in lambic do not seem to have the ability to produce beta-glucosidase nor utilize cellobiose. Daenen et al. (2007) found that none of the B. bruxellensis strains isolated from lambic could utilize cellobiose, but strains of B. anomalus and B. custersianus isolated from lambic could utilize cellobiose . Additionally, a study by Tyrawa et al. from Escarpment Laboratories agreed that wine isolated strains were generally better at fermenting cellobiose than strains isolated from beer at 15°C (59°F). However, at 22.5°C (72.5°F) most of the beer strains started to utilize cellobiose after a few days of incubation (they preferred other food sources such as glucose and maltose), indicating that temperature plays a role in whether Brettanomyces can ferment certain sugars , and the table from the NCYC Brettanomyces strains suggests that fermenting cellobiose is generally rare for B. bruxellensis. This also suggests that not only are B. bruxellensis strains that are isolated from beer generally unable to break down glycosides, but they are probably also unable to utilize the cellobiose in wooden barrels as a food source (although higher temperature might allow some beer strains to start fermenting cellobiose).

See the Glycosides page for more details.

Hop Biotransformation

Colomer et al. (2020) was the first to examine the effects of Brettanomyces on hops during fermentation. The researchers selected 4 strains of B. bruxellensis and 1 strain of B. anomalus that had the genetic markers for producing beta-glucosidase enzyme, and fermented them as a primary fermenter in a non-dry hopped beer, and also performed a second experiment where they inoculated the Brettanomyces strains in a dry hopped beer that was first fermented with S. cerevisiae. Monoterpene alcohols were measured before and after inoculation with the 5 strains of Brettanomyces. In both beers, they found a decrease in geraniol and a rise in beta-citronellol after the inoculation with Brettanomyces. Beta-citranellol reached a level of 31.5 μg/L with one of the strains, which is a much higher level of beta-citronellol than anything that has been reported with S. cerevisiae, suggesting that some strains of Brettanomyces might be better at converting monoterpenes from hops than Saccharomyces. See Figure 5 in the open access study. Interestingly, the strains with the highest beta-glucosidase activity produced the lowest amount of beta-citranellol, indicating that there is no link between beta-glucosidase activity in Brettanomyces and hop biotransformation. This might be due to the fact that most of the beta-glucosidase enzyme is produced within the cell and is not released outside of it. The researchers hypothesized that this conversion could be due to two proteins referred to as BbHye2 and BbHye3 that can be present in Brettanomyces metabolism .

For more information on glycosides, see the Glycosides page. For more information on hop biotransformation in general, see the Hops page.

Nitrogen Metabolism

Other than sugars, nitrogen in the form of amino acids is an essential nutrient for yeast. One significant source for nitrogen in wort is boiling hops and dry hopping . Brettanomyces can survive in environments that are very low in nitrogen, with one report being as low as 6 mg N/L of yeast assimilable nitrogen (YAN) which is less than most finished wines contain . 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. Preferred sources of nitrogen include the amino acids glutamine (aerobically and anaerobically), glutamate and aspartate (anaerobically) , as well as possible secondary sources such as lysine, histidine, arginine, asparagine, aspartic cid, glutamic acid, and alanine . Some strains of Brettanomyces can metabolize other nitrogen sources, such as the amino acids proline and arginine . 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) . Other compounds from Saccharomyces autolysis may also be used by Brettanomyces, such as glucose, fatty acids, nucleotides, polysaccharides, polypeptides, and other proteins . 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 .

Secondary Metabolites

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

Ester Production

Brettanomyces is capable of synthesizing several ethyl esters from ethanol and fatty acids, as well as other types of esters from various alcohol types (methanol, for example). Among the most prolific of these are ethyl acetate (synthesized from ethanol and acetic acid), ethyl lactate (synthesized from ethanol and lactic acid), phenethyl acetate, ethyl caproate, ethyl caprylate, ethyl deconoate , along with the hydrolysis (breakdown) of isoamyl acetate. Esters have been found to attract fruit flies and other flying insects, which help many species of yeast transfer from one food source to another (namely 2-phenyl-ethanol, 3-methyl-1-butanol, ethyl acetate, 2-methyl-1-butanol, and 3-methyl-3-butenol). Some of these esters are also released by blooming flowers and it is thought that the attraction to flowers by insects is also driven by these same esters . During non-mixed fermentations where lactic acid is minimal to none, insignificant amounts of ethyl lactate esters are produced, whereas ethyl caprylate and ethyl caproate have a general increase. With the addition of lactic acid, ethyl lactate levels are greatly increased although may still not reach the flavor threshold level of 250 mg/L (strain dependent), and ethyl acetate is generally slightly increased. The amounts of esters produced vary widely based on species and strain . 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 .

Ester production peaks towards the end of growth and is influenced by temperature, aeration/agitation, and pH. Spaepen and Verachtert found in one study that the optimal temperature for growth and thus ester production was 28°C (77°F), although they did not test higher temperatures. This study also found that continuously shaken samples produced relatively fewer esters, as well as samples that were not exposed to oxygen at all. The highest ester production was found under conditions of limited oxygen supply (semi-aerobic versus aerobic or anaerobic), 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 . Another study by Tyrawa et al. found that all strains of B. bruxellensis tested produced above threshold levels of ethyl caproate, ethyl caprylate, and ethyl deconoate esters at 15°C versus 22.5°C, but for some strains the higher fermentation temperature of 22.5°C produced significantly more of these esters than the lower 15°C temperature (other strains produced similar levels of esters at both temperatures, although they fermented slower at 15°C) .

Pitching rate of Brettanomyces may have a slight effect on ester production levels, but the differences caused by pitching rate probably do not have a significant impact on the sensory character of the beer . 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) . The aromatic amino acids phenylalanine, tryptophan, and tyrosine have been associated with higher ester formation .

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

See also:

• Brettanomyces secondary fermentation experiment.
• Understanding Esterification, Sour Beer Blog.

Ester	Precursors	Flavor/Odor Threshold	Molecular Formula	Notes
Amyl octanoate (spicy, clove, chemical, plastic)	Amyl alcohol and caprylic acid		C13H26O2	Also known as pentyl octanoate, it is a flavoring agent .
Ethyl acetate (fruity, pineapple, pear, solventy, nail polish remover)	Acetic Acid and ethanol	33ppm (odor), 100ppm (flavor)	C4H8O2	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 .
Ethyl butyrate (pineapple, mango, tropical fruit , juicy fruit gum )	Butyric Acid and ethanol	0.4ppm (flavor)	C6H12O2	Low levels of production by some species of Brettanomyces; production decreases with higher acidity . Also known as ethyl butanoate .
Ethyl caproate (sweet, fruity, pineapple, banana, apple or aniseed)	Caproic acid and ethanol	0.2ppm (flavor)	C8H16O2	Also known as Ethyl hexanoate, Ethyl butyl acetate, and butylacetate . Can also be produced by Saccharomyces species .
Ethyl caprylate (Sweet, waxy, fruity and pineapple with creamy, fatty, mushroom and cognac notes )	Caprylic acid (contained in buckwheat; produced by yeast autolysis) and ethanol	15ppb (flavor)	C10H20O2	Also known as ethyl octanoate .
Ethyl Decanoate/Ethyl Caprate (brandy, fruity, oily, grape)	Decanoic acid (Capric Acid) and ethanol	0.5ppm (flavor in water)	C12H24O2	Also known as Ethyl caprate, Ethyl caprinate, and Capric acid ethyl ester . Can also be produced by Saccharomyces species >
Ethyl hexanoate (Apple or aniseed )	Hexanoic acid and ethanol .	0.2ppm (flavor in beer)	C8H16O2	Also produced by both ale and lager yeast; it is a key flavor note in many beers . High amounts Produced by two strains out of 9 B. bruxellensis in one study .
Ethyl isobutyrate (Pungent, etherial and fruity with a rum-and egg nog-like nuance )	Isobutyric acid and ethanol	0.1 ppb (odor in water)	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 . Produced in significant amounts by 3 out of 9 strains of B. bruxellensis tested in one study .
Ethyl isovalerate (fruity, sweet, berry-like with a ripe, pulpy fruit nuance, artificial grape )	Isovaleric Acid and ethanol	30ppm (flavor)	C7H14O2 (same as ethyl valerate)	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 . Also known as Ethyl 3-methylbutanoate . Not identified as a major product of B. bruxellensis, but is produced in large quantities by some strains .
Ethyl lactate (fruity, creamy, rum )	Lactic Acid and ethanol	0.2 ppm-1.66 ppm (odor)	C5H10O3	Increases production with higher amounts of Lactic Acid
Ethyl valerate (Sweet, fruity, acidic, pineapple, apple, green, berry, tropical, bubblegum )	Valeric Acid (pentanoic acid) and ethanol	1500-5000 ppm (odor)	C7H14O2	Valeric acid quantities found in beer are minimal (0-1 ppm) and below odor threshold , and is probably also the case for Ethyl valerate. Ethyl valerate is also known as ethyl pentanoate . Also found in apples, bananas, guava, stawberry, cheeses, rum, whiskey, cider, sherry, grape wines, cocoa, coffee, honey, and passion fruit . Not identified as a major product of B. bruxellensis, but is produced in large quantities by some strains .
Ethyl-2-methyl butyrate (minty, menthol, citrus, green apple)	Ethanol, methanol, and butyric acid		C7H14O2	Also known as ethyl 2-methylbutanoate . Found in bilberry, and in many other fruits, e.g.raw and cooked apple, apricot, orange, grapefruit. Used as a fruit flavor additive . Identified as a major product of B. bruxellensis .
Isoamyl acetate (banana)	Acetic Acid and Isoamyl alcohol	1.1ppm (flavor)	C7H14O2	Produced by certain Saccharomyces strains but concentrations are generally reduced by Brettanomyces. Brettanomyces produces only very small amounts itself
Octyl butyrate (spicy, eucalyptus, floral, plastic)	Octanol and butyric acid		C12H24O2
Pentyl formate (artificial fruit, candy, chemical)	Pentanol and formic acid		C6H12O2	Also known as amyl formate .
Phenethyl acetate (sweet, honey, rose flower like)	Acetyl-CoA and 2-phenylethanol	3-5ppm (odor), 5-10ppm (flavor)	C10H12O2	Produced in very small amounts in Lambic . Can also be produced by Saccharomyces species
Phenethyl formate (artificial floral, perfume, wild flower, solvent)	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 through the decarboxylation of hydroxycinnamic acids (HCAs) by yeast, and also in small amounts by long boils with a portion of the wort coming from wheat (3+ hours resulted in 0.3 ppm of 4VG). HCAs, such as ferulic acid and p-coumaric acid, are present in the non-starch polysaccharide arabinoxylan in malted barley and wheat. They are released into wort during mashing at levels that are far below their flavor thresholds (greater than 500ppm flavor threshold) . Some strains of Oenococcus oeni and Lactobacillus, as well as some strains of yeast such as Pichia spp, have been found to produce HCA's via cinnamoyl esterase activity in wine, although when these strains have been used in wine to increase the HCA levels, the final phenol levels produced by Brettanomyces were the same as wine that did not have an increase in HCA levels (the precursors in wine that lead to HCA's are different than they are in beer) . The esters in grape must that contain HCA's (ethyl ferulate and ethyl coumarate) can also be formed by acidic hydrolysis which occurs at the low pH of wine, and HCA's can then be released from these esters. This formation of esters is a slow process in wine, with one study reporting ~0.03 ppm of ethyl ferulate and ~0.4 ppm of ethyl coumarate at the end of primary fermentation and ~0.09 ppm of ethyl ferulate and ~1.4 ppm of ethyl coumarate after 10 months of barrel aging . We are not aware of any studies that have reported an increase in HCA's from acidic hydrolysis over time in beer; however, this is a standard laboratory technique for forcing the release of HCA's from barley (although this lab technique uses a lower pH then that of sour beer). In addition, it has been demonstrated that spent yeast (S. cerevisiae collected after beer fermentation) contains a small fraction of phenols and polyphenols absorbed from wort during fermentation . It is therefore conceivable that HCA levels could increase in sour beer over time.

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 , Brettanomyces is also able to reduce these vinyl phenol derivatives to ethyl phenol derivatives. Phenolic acid decarboxylase (PAD) is the enzyme that converts the HCAs into vinyl phenols. Vinyl reductase (VA) is the enzyme that reduces vinyl phenols to ethyl phenols . Phenol production has been observed to occur shortly after inoculation of Brettanomyces and has been hypothesized to play a large role in replenishing NAD⁺ to alleviate the initial lag growth phase in Brettanomyces . While almost all strains of Brettanomyces produce ethyl phenols, one strain of Brettanomyces anomalus has been found that has lost the genetic capability to produce phenols .

These vinyl derivatives have similar tastes to the ethyl derivatives but have lower flavor thresholds. Levels of all compounds produced vary depending on species and strain of Brettanomyces. Although the production of ethyl phenols has been identified to occur higher in substrates with more available sugars, and this has also correlated with higher growth , some data supports that pitching rate does not have an effect on how much phenol content is produced by Brettanomyces. Additionally, Curtin et al. (2013) showed that while both cell growth and attenuation was inhibited in anaerobic conditions in wine, phenol production was not (in fact, the phenol production was inhibited by aerobic conditions). They also showed that each of the three strains of B. bruxellensis tested all produced the same amount of phenols, while other flavor compounds such as esters were produced at different levels by each of the strains . Riley Humbert's Bachelors thesis also reported no correlation between fermentation rate and phenol production in several strains of B. bruxellensis . Perhaps growth itself is not as much of a factor in producing phenols, but having sugars available for metabolism is. This contradicts the somewhat popular belief that under-pitching Brettanomyces produces more "funky" flavors. Additionally, perhaps some strains are perceived as "funkier" than others due to less ester production and more fatty acid production (isobutyric acid, for example), rather than more phenol production.

Another study by Tyrawa et al. found that fermentation temperature also did not have a significant effect on phenol production in 9 strains of B. bruxellensis. Given the same wort composition, strains of B. bruxellensis produced similar levels of phenols at both 15°C and 22.5°C. The ester production was affected by this temperature difference in some strains but not others (see Esters above). Assuming that phenols contribute the "funky" flavor characteristics, this suggests that perhaps a lower balance of esters to phenols produces a more "funky" tasting beer more so than a beer with more phenol content. If so, a lower fermentation temperature may be one way to emphasize phenols over fruity esters . Both Tyrawa and Humbert reported that there was no correlation between flavor profiles from phenol production of different strains of Brettanomyces bruxellensis and their origin .

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

It has been demonstrated in wine that some phenols can be masked by other flavor compounds, especially lower levels of phenols. Schmuaker et al. (2018) showed that wines that were spiked with 4EG, 4EP, and isobutyric acid were preferred more when additionally spiked with whiskey lactone (oaky flavor). The oaky flavor at least partially masked the perception of phenols on the palate . It has been hypothesized by some members of Milk The Funk that higher esters could also mask the perception of phenols in beer (see 100% Brettanomyces fermentation).

See also:

• Effects of Saccharomyces Strain Selection and Staggered vs Co-Pitch
• Brettanomyces secondary fermentation experiment.
• See the Phenol Explorer website for more information on sources of precursors.
• Food sources of Hydroxycinnamic acids (p-Coumaric acid, ferulic acid, caffeic acid, etc.).
• Brü Lab Podcast Episode 030 | Phenolic Compounds In Beer w/ Dr. Mike Lentz.

Phenol	Phenol Type	Precursors	Flavor/Odor Threshold	Molecular Structure	Notes
4-Vinylphenol (Musty, Medicinal, Band-aid, Plastic)	Vinyl phenol	p-Coumaric Acid	0.2 ppm (flavor; in beer)	C8H8O	Production level is different across species/strains of Brettanomyces . Coumaric acid levels vary greatly between barley varieties; for example, between 320 µg/kg to 950 µg/kg in different varities of barley husks and 73 µg/kg to 657 µg/kg in different varities of barley malt . Coumaric levels are generally higher in barley malt than they are in wheat malt. Coumaric acid is stable through the wort boiling process .
4-Vinylguaiacol (Clove)	Vinyl phenol	Ferulic Acid	0.3 ppm (flavor; in beer)	C9H10O2. Also known as 2-methoxy-4-vinyl phenol .	Produced by some strains of S. cerevisiae (see Saccharomyces) . Some Brettanomyces species/strains may also be able to produce this compound at varying levels . Organic malts have been linked to higher levels of 4VG, vanillan, and their malt precursor ferulic acid . Ferulic acid is released during mashing, with an optimal mash temperature of 40-45°C (104-113°F) and a mash pH of 5.7-5.8 (enyzmatic release of ferulic acid is optimal at a pH of 7.5, but this high of a pH is difficult to achieve during mashing and would cause other enzymatic problems during the later steps of the mash) . Some studies have found that ferulic acid is generally more efficiently extracted from a combination of 70% barley malt and 30% wheat malt (not raw wheat), despite studies showing that barley malt often contains more ferulic acid than wheat malt (see this MTF thread that explains why this is) . A more recent studies disagreed and found a linear increase soluble ferulic acid correlated with higher percentages of wheat malt . Malting parameters also affect the levels of ferulic acid in malt; for example, wheat malt with higher germination temperatures (24-26°C versus 12-18°C) were shown to form more ferulic acid in one study that looked at the impact of germination temperature and aeration during germination of barley and wheat malt . Ferulic acid is also There is also a correlation between how dark a malt is (or how highly kilned it is and how much melanoidin content it has) and how much ferulic acid the malt has; the darker the malt, the more ferulic acid (however, roasted malts were not tested in the referenced study) . Ferulic acid is stable through the wort boiling process .
4-Vinylcatechol (Plastic, Bitter, Smokey)	Vinyl phenol	Caffeic Acid		C8H8O2	Production level is difference across species/strains of Brettanomyces .
4-Ethylphenol (Barnyard, Horsey, Spicy, Smoky, Medicinal, Band-Aid )	Ethyl phenol	4-vinylphenol	0.3 ppm (odor; in beer)	C8H10O	Also known as 1-Ethyl-4-hydroxybenzene and P-Ethylphenol . Identified as a major product of B. bruxellensis . Richard Preiss of Escarpment Laboratories describes pure 4EP as the following, "barnyardy with a slight solvent edge at low concentrations, and full on hospital antiseptic/bandaid/barnyard at high concentrations. Really quite complex, but maybe not in a good way."
4-Ethylguaiacol (Smokey, Spicy, Clove )	Ethyl phenol	4-vinylguaiacol	0.13 ppm (odor; in beer)	C9H12O2	Also known as 4-Ethyl-2-methoxyphenol . Identified as a major product of B. bruxellensis . Richard Preiss of Escarpment Laboratories describes pure 4EP as the following, "4EG is a lot more pleasant (than 4EP), very light woodsmoke at low concentrations and heavier smoke and vanilla-like (actually "tonka bean" is closer but not as universally understood) at high concentrations."
4-Ethylcatechol (Band‐aide, Medicinal, Barnyard)	Ethyl phenol	4-Vinylcatechol		C8H10O2	Also known as 4-ethylbenzene-1,2-diol .

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 and alcohol, Brettanomyces species produce acetic acid as a byproduct of glucose fermentation. The more oxygen that is present, the more acetic acid is produced and the less ethanol is produced by Brettanomyces . In an environment with oxygen present, sugar is reduced to pyruvate within the cell and is then broken down into acetaldehyde which is then enzymatically oxidized into acetic acid or ethanol (dubbed the Custers effect). 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) . 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 species and strains, and it is limited by limiting oxygen exposure (see aging mixed fermentation beer for practical tips on limiting oxygen exposure). For example, B. naardenensis and B. custersianus produce less acetic acid than other species of Brettanomyces . 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) . 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. Some strains can also produce succinic acid as a byproduct of fermentation under semi-aerobic conditions, but not anaerobic conditions .

The production of acetic acid can vary between species of Brettanomyces. For example, it has been reported that in the presence of oxygen B. naardenensis produces less acetic acid than B. bruxellensis .

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

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 . 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 .
Butyric Acid (Vomit, bile)	Associated with supplements of phenylalanine or tyrosine	Fatty acid.
Capric acid (Barnyard animal odor/taste)		Fatty acid. Also found in milk, coconut oil, and seed oils . Also referred to as Decanoic acid .
Caproic acid (Fatty, cheesy, waxy, barnyardy)		Fatty acid. Also known as hexanoic acid .
Caprylic acid (Rancid-like smell and taste		Fatty acid. Also found in milk. Gives a waxy/oily mouthfeel. Flavor is more intense at low pH levels. Also called octanoic acid.
Heptanoic acid (sweaty, solvent, rotten, barnyard)	Associated with phenylalanine or tyrosine	Fatty acid. Also known as enanthic acid .
Isovaleric Acid (Feety, parmesan)	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)		Fatty acid. Also known as dodecanoic acid and dodecylic acid .
Nonanoic acid (Rancid odor)		Fatty acid. Also known as pelargonic acid .
Succinic acid		Produced by some strains of b. bruxellensis under semi-aerobic conditions (small amounts of available oxygen), but not anaerobic (no available oxygen), and only during the stationary phase (after growth has finished) .
Undecanoic acid (coconut, balsam, oily, vanilla)	Associated with tyrosine .	Fatty acid. Also known as undecylic acid .

Biogenic Amines

Biogenic amines are produced by all living things and are present in many fermented beverages. High dosages can lead to health issues such as vomiting, headache, asthma, hypotension, and cardiac palpitation. Thus, biogenic amines have been studied intensely. Levels of histamine below 50 mg/kg in the US and 400 mg/kg in the UK are considered safe for human consumption (these levels are usually regulated for meets and fish; see reference) . For more information, see "Fact or Fiction – Biogenic Amines in Beer" by Dr. Bryan Heit.

The production of biogenic amines by Brettanomyces is strain dependent. Relatively low levels of biogenic amines can be produced by various strains. Vigentini et al. (2008) found that 5 strains of B. bruxellensis were able to produce detectable levels of putrescine, cadaverine and spermidine. Most strains produced 0.4 mg/L or below of these biogenic amines, while one strain produced 1.2-1.3 mg/L of spermidine at 60 days, which reduced 0.70-0.85 mg/L at 95 days. In general, the maximum biogenic amines were produced between 40-60 days .

Agnolucci et al. (2009) found similar results with 7 strains of B. bruxellensis. None of the strains produced histamine, synephrine, tyramine or spermine. All of the strains produced under 1 mg/L of most biogenic amines, with hexylamine being produced more so in the range of 1.02-3.92 mg/L :

Source: Agnolucci M, Vigentini I, Capurso G, Merico A, Tirelli A, Compagno C, Foschino R, Nuti M (2009) Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines. Int J Food Microbiol 130:238–244

Filipe-Ribeiro et al. (2019) used a statistical model to look for a correlation between biogenic amine levels and the presence of Brettanomyces in different vintages of Portuguese wines. They did this by looking at the levels of ethyl phenols in the wines, which is a strong indicator of the presence of Brettanomyces. They found no statistical evidence that the presence of Brettanomyces in wine had any effect on the levels of biogenic amines, positive or negative .

For more information on biogenic amines, see the following:

• Biogenic amines in spontaneous fermentation.
• Biogenic amine production in Pediococcus.
• "Spontaneous fermentation and biogenic amines" by Dr. Dave Janssen; review of several studies that looked at the levels of biogenic amines in different beers and their production, and their potential flavor contribution.
• "Fact or Fiction – Biogenic Amines in Beer" by Dr. Bryan Heit; an analysis of biogenic amines in spontaneously fermented beer and associated health concerns.

Glycerol

Brettanomyces is known for not producing much glycerol in beer. Glycerol is a colorless, sweet-tasting, viscous liquid that is thought to be an important contributor to the mouthfeel of beer. Glycerol is produced as a stress response by a wide range of microbes, including S. cerevisiae, and various species and strains of Debaryomyces, Candida, Lachancea, and Zygosaccharomyces. Despite not producing amounts of glycerol that are perceivable in beer, some strains of Brettanomyces bruxellensis actually produce glycerol which is stored inside of their cells as a response to osmotic stress. They can also uptake glycerol into their cells. Doing so allows the cells to survive osmotic pressure . It is currently not known how many strains are capable of producing glycerol internally, or if this amount of glycerol has any impact on perceived mouthfeel of a beer if a substantial amount of Brettanomyces cells eventually autolyze (see this MTF thread). The role of glycerol in creating mouthfeel is debatable in the wine world .

Other Compounds

Compound Produced	Chemical Type	Precursors	Threshold	Notes
2-Methoxyphenethyl alcohol (white glue, plastic, mimeograph sheet)	Alcohol	Associated with ferulic acid
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 .
2-Methyl-1-butanol (oxidized/canned fruit, plastic, sulfur)	Alcohol	Associated with ferulic acid, phenylalanine, and tyrosine .		Identified as a major product of B. bruxellensis .
Bisabolene (spicy, tropical, toasty, wood resin, minty)	Terpene	Associated with caffeic acid .
Decanol	Alcohol			Also known as decyl alcohol .
Isoamyl alcohol	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 . Commonly produced by many strains of B. bruxellensis. Identified as a major product of B. bruxellensis .
Nerilidol	Terpene			Often found in bitter gourd and is a component of many essential oils. It is often used as a flavoring agent .
Nonanal (lemon furniture polish, air fresener, oily	Aldehyde	Associated with tyrosine
Ocimene (sweet floral, hyacinth, jasmine)	Terpene	Associated with phenylalanine
Octanol	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 .
Phenethyl alcohol (floral, dried rose )	Alcohol			Also known as phenethanol. Identified as a major product of B. bruxellensis .
Phenylacetaldehyde (honey, floral rose, swaat, powdery, chocolate with a slight earthy nuance )	Aldehyde
Tetrahydropyridine (Cheerios®, mousy, urine, cracker biscuit, corn chips)	Ketone	L-Lysine, ethanol, and oxygen	Varies	See the Tetrahydropyridine page for more details.
Hydrogen sulfide (rotten egg)	Chalcogen hydride gas		4 µg/l in beer	Produced in high amounts by B. custersianus and B. naardenensis .
Diacetyl/butanedione (butter)	Vicinal diketone		0.1–0.2 ppm in lager and 0.1–0.4 ppm in ales, although flavor thresholds as low as 17 ppb and 14–61 ppb have been reported .	Produced by all strains and all species of Brettanomyces except B. naardenensis; the amount that it is produced varies widely and not much is known about what determines diacetyl levels produced from Brettanomyces . Brettanomyces generally produce very low amounts of diacetyl (0.019 - 0.048 mg/L). It is hypothesized that Brettanomyces can reduce diacetyl during its maturation phase in a similar way to Saccharomyces species, but this has not been investigated that we are aware of. It has been reported that diacetyl reduction is faster at a lower pH of around 3.5, which is a typical pH range for sour beer and might be one of the contributing factors to a lack of anecdotal reports of diacetyl in sour beer .

Commercial Cultures

Pure cultures. In cooperation with Funk Factory.

Bootleg Biology

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	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. Single isolate .	West Flanders, Belgium brewery specializing in funky, sour, mixed-fermentation beers.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	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. Single isolate .	Family-owned brewery producing Gueuze in West Flanders, Belgium.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	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. Single isolate .	Dry-hopped, Unblended Lambic Produced by a Traditional Lambic Brewery in Brussels, Belgium.

Brewing Science Institute

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	Brettanomyces bruxellensis	Medium intensity Brett character. Classic strain used in secondary fermentation for Belgian style beers and lambics.	Thought to be the same as White Labs WLP648; however, side by side sensory comparisons seem to indicate that they might not be the same . Commercial pitches only.
Claussenii	Dekkera anomala	Brettanomyces anomalus	Brettanomyces clausenii	Low intensity Brett character.	Thought to be the same as White Labs. Commercial pitches only.
CMY1	Dekkera bruxellensis	Brettanomyces bruxellensis	bsi		Chad Yakobson's mutation of BSI Drie. Commercial pitches only.
Drie	Dekkera bruxellensis	Brettanomyces bruxellensis	Brettanomyces bruxellensis var. Drei	Highly aromatic of tropical fruit come with aging. Works well with citrus and fruity hops.	Isolate from Drie Fonteinen; Pro-Brewers only. Recommended fermentation levels to get the most character out of Drie include 75-77°F . Commercial pitches only.
Lambicus	Dekkera bruxellensis	Brettanomyces bruxellensis	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.	Commercial pitches only.

Craft Cultures

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Unknown	Unknown	Unknown	CCYL410 Razz Brettanomyces™	Great for use in secondary fermentation for Belgian style beers and lambic style beers.	Isolated from a wild rasberry in Michigan's Upper Peninsula along the shore of the Portage Lake near Houghton Michigan . Commercial pitches only.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	CCYL411 Brettanomyces bruxellensis	Exhibits medium intensity Brett character .	Commercial pitches only.
Claussenii	Dekkera anomala	Brettanomyces anomalus	CCYL412 Brettanomyces claussenii	Low intensity Brett character. The Brett flavors produced are more subtle than CCYL410. More aroma than flavor contribution. Fruity, pineapple like aroma .	75-85% attenuation, low flocculation, 8-12% alcohol tolerance, 67-74°F fermentation temperature . Commercial pitches only.
Lambicus	Dekkera bruxellensis	Brettanomyces bruxellensis	CCYL413 Brettanomyces lambicus	High intensity Brett character. Defines the "Brett character": Smoky and spicy flavors. This strain is often found in Lambic, Flanders, and sour brown style beers .	Commercial pitches only.

East Coast Yeast

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Anomala	Dekkera anomala	Brettanomyces anomalus	ECY-04	This species displays strong esters of juicy-fruit gum, moderate funk and gradual acidity over time. A super-attenuator, up to 100%	beer - Adelaide, Australia
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	ECY-05	funky with barnyard notes accompanied by some fruit. May no longer be available .	isolated from Belgian stout.
Nanus	Brettanomyces nanus	Brettanomyces nanus	ECY-16	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
Custersianus	Brettanomyces custersianus	Brettanomyces custersianus	ECY-19	Light fruit and hay. May no longer be available .	Bantu beer brewery, South Africa
Naardenensis	Brettanomyces naardenensis	Brettanomyces naardenensis	ECY-30	Strawberry, honey, ripe fruit with a tart, citrusy acidity after 6mo of aging. Creates an abundance of acidity quickly and may initially display a mousy-like tainted flavor but dissipates over time leading to ester production with hints of strawberry. Best used with Saccharomyces; aging up to 6 months is recommended .	Isolated from Dr. Pepper
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	ECY35	Funky with barnyard notes accompanied by ripe tropical fruit .	Isolated from bottle of Belgian gueuze.
Anomala	Dekkera anomala	Brettanomyces anomalus	ECY36	Notes of pineapple esters with acidity over time, no funk or barnyard present. Versatile .
Lambicus	Dekkera bruxellensis	Brettanomyces bruxellensis	ECY37	Cherry-stone and strong barnyard .

Escarpment Laboratories

• "How to Choose a Brett Strain," a guide to Escarpment Laboratories Brettanomyces products on their blog.

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces 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 .	Isolated from a bottle of Orval .
Claussenii	Dekkera anomala	Brettanomyces anomalus	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. Potentially no longer available (no longer listed on website) .
Bruxellensis	Dekkera bruxellensis	Brettanomyces 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 . See this MTF thread on experiences using it for 100% fermentation.
Unknown	Unknown	Unknown	Brett M	This strain offers balanced funk fast. Suited for use in sour beers, saisons, and other barrel-aged treats .	Michigan
Bruxellensis	Dekkera bruxellensis	Brettanomyces 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 .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	Brussels Brett	This Brettanomyces bruxellensis strain displays a balanced selection of fruity characteristics, with testers noting plum, red berry, citrus, and red apple, alongside subtle acidity. It is suitable for primary or secondary fermentation, but does shine in secondary with extended aging, where it displays prominent funky flavours. Attenuation: 80+% // Optimum Temp: 22-25ºC // Alcohol tolerance: 12%+ // Flocculation: Medium-low .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	Berliner Brett II	Isolated from a bottle of Schultheiss Berliner Weisse, this B. bruxellensis strain produces notes of orchard fruit and pineapple. Complements Berliner Brett I nicely to produce a complete classic Berliner profile .

Fermmento Labs (Brazil - CLOSED)

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	FB2 Heavy Funkies

Imperial Organic Yeast

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

Inland Island Yeast Laboratories

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	INIS-901 Brettanomyces bruxellensis I	Isolated from a brewery in Brussels this particular Brettanomyces strain is 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	Brettanomyces bruxellensis	INISBC-902 Brettanomyces bruxellensis II	This strain is capable of fermenting a beer without the use of a Saccharomyces cerevisiae. 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	Brettanomyces 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
Unknown	Unknown	Unknown	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	Brettanomyces bruxellensis	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	Brettanomyces anomalus	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 .

GigaYeast

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces 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	Brettanomyces 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	Brettanomyces bruxellensis	Sweet Flemish Brett GB144	Produces a sweet, slightly fruity profile with just a hint of barnyard and spicy phenolics

Jasper Yeast

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	JY033 - Brett brux Amsterdam	Slow growing, good for conditioning in the style of Orval	Amsterdam, Netherlands
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	JY044 - Brett brux Rosa	Comparable to JY033, but slightly more funky, tart and aggressive.	California, USA
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	JY061 - Brett brux Chateaux	JY87 is a Brettanomyces yeast isolated by Jasper Yeast 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.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	JY062 - Brett brux Abbey	Mild aromatics and phenolics, but quite tart and high tendency to produce acetic acid in the presence of oxygen.	California, USA
Lambicus	Dekkera bruxellensis	Brettanomyces bruxellensis	JY157 - Brett brux lambicus	Good for Brettanomyces beers where an intense character is preferred. Prolonged aging can produce almost a cherry like sourness. Optimum temperature: 80-90°F (27-32°C)	Belgium
Claussenii	Dekkera anomala	Brettanomyces anomalus	JY196 - Brett c	Optimum temperature: 80-90°F (27-32°C). Pineapple aroma, but can also develop a leather character. This yeast is not suitable to be used on its own in malt based worts because of its limited fermentation capacity.	Belgium

Mainiacal Yeast (CLOSED)

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MYOrval	Depending on how this strain will dictate its outcome. In primary fermentation, it will lend stone fruit notes and a very thin mouthfeel. Although in secondary it can produce cherry notes all the way to barnyard like "funk". We find to obtain this funk quality you will need a decent amount of aged hops in the boil alongside a secondary fermentation with this strain.	Isolated from a 5 year old bottle of Orval. 65-80°F fermentation temperature with 65-100% attenuation . Commercial pitches only; occasionally available to homebrewers.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MYLBB1	It produces a stone fruit forward Brett quality when used in primary fermentation and like many Bretts it does not produce high amounts of glycerol so mouthfeel can be expected to be thin. Used in secondary it can lend notes of cherries and a light barnyard like aroma.	isolated from a red wine barrel that was used by a winemaker. 65-80°F fermentation temperature with 65-100% attenuation . Commercial pitches only.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MYLBB2	Depending on how this strain is used will determine the outcome. In primary fermentation it produces light stone fruits with a hint of barnyard like hay. You can also expect a thin mouthfeel due to the low glycerol production. In secondary it will produce a more heavy barnyard like aroma with hints of cherries and raspberries.	American Spontaneous Producer, isolated from a bottle. 60-80°F fermentation temperature with 75-95% attenuation . Commercial pitches only.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MYLBB3	This strain of Brett b is better suited for either co-fermentation with a Sacc strain or used in secondary. If used for primary fermentation it can produce significant sulfur like aromas. This strain can produce a significant amount of funk if using aged hops at a similar rate as traditional Lambics. It also produces notes of pineapples.	Isolated from a Belgian spontaneous producer. 55-78°F fermentation temperature with 75-100% attenuation . Commercial pitches only.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MYLBB4	This Brett strain is described as 'unique'. It's not great for primary fermentation, especially in kettle sours as it throws very odd smells off. However, it works better as a secondary Brett strain producing peach and light fresh apple notes, sometimes also producing a spice character.	Isolated from an apple orchard in Maine. 50-80°F fermentation temperature with 70-90% attenuation . Commercial pitches only.

Omega Yeast Labs

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Claussenii	Dekkera anomala	Brettanomyces anomalus	Brettanomyces claussenii OYL-201	Contributes more Brett aroma than flavor. Fruity, pineapple-like aroma. Flocculation: low, Attenuation: 70-85%, Temp: >85°F, Alcohol Tolerance: medium-high, compares to WLP645. Can ferment lactose . Pro brewers only. Recommended to use in conjunction with brewers yeast; not recommended for 100% Brettanomyces fermentation as it doesn't attenuate wort on its own very well .
Bruxellensis	Dekkera bruxellensis	Brettanomyces 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	Brettanomyces bruxellensis	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.
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	Brettanomyces bruxellensis OYL-216	White wine character and light funk, and develops its character rather quickly. Brett character will be apparent within a few weeks of reaching terminal gravity and will continue to develop if given additional conditioning time. Flocculation: low, Attenuation: 70-85%, Temp: 68-80°F, Alcohol Tolerance: medium-high. Potentially the same B. bruxellensis strain as their C2C blend . Pro brewers only.	A US Northwest brewery.

Propagate Lab

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MIP-701 Brett Brux I	Used for secondary fermentation in Belgian-style beers such as lambics, this strain creates a medium-intensity, earth-forward character in finished beer. A historic brewery in Belgium uses this yeast in secondary fermentation and bottling to produce the signature flavor of its beer .	(Editor's note: this is likely to be a strain isolated from Orval).
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MIP-702 Brett Brux II	a strain used for secondary fermentation in Belgian-style beers such as lambics. It creates a medium-intensity, earth-forward character in finished beer. Balanced between barnyard and fruit .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	MIP-703 Brett Brux III	A strain used for secondary fermentation in Belgian-style beers such as lambics. Characterized as "fruity with tropical fruit dominating the aroma" .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	YH169	In collaboration with Wild Pitch Yeast. High attenuation, produces a lemony/tart/subtle “Brett” aroma and flavors characterized as lemon, limoncello, and mint with a slight undertone of the more typical Brett barnyard funk. Can be used at a wide variety of temperatures (68-99 F) with a potential flavor sweet spot of 80-85 F. Would work well in a saison or Brett IPA .	Isolated from a spontaneous fermentation in Indianapolis, IN.
Unknown	Unknown	Unknown	MIP-710 Brett Stave I	strain used for secondary fermentation in Belgian-style beers such as lambics; characterized as "fruity".	This particular strain was isolated from a Colorado Brewery and produces intense fruit notes .
Unknown	Unknown	Unknown	MIP-714 Brett Stave IV	Produces barnyard like aromas and is a very aggressive strain.	This strain was isolated from a bottle of beer originally produced in the Netherlands .
Clausenii	Dekkera anomala	Brettanomyces anomalus	MIP-720 Brett clausenii	A strain used for secondary fermentation in Belgian-style ales and English Old Ales. The yeast can be fairly neutral in aroma and works well by itself. When it is paired with a phenol producing yeast it will create barnyard like aromas .
Unknown	Unknown	Unknown	MIP-750 Brett tool	An aggressive strain that produces strong barnyard aroma. Works well as a blend.	Isolated from a bottle of European beer fermented with fruit .
Unknown	Unknown	Unknown	MIP-760 Brett phantom	A highly fruity strain that will ferment well by itself or pitched into a blend.	Isolated from a famous saison producer .
Unknown	Unknown	Unknown	BTN-70 Feints	Characterized as sweet tarts, ripe peach skin, clementine, white grapes, and a hint of candied strawberry with a backbone of soft Brett funk.	Isolated from a natural wine from Mendocino County, California .
Unknown	Unknown	Unknown	BTN-81 Yellow Jacket	Characterized as Brett-forward with notes of sweet tarts, ripe peach skin, dried lime peel, tangerine pith.	Isolated from a yellow jacket insect .

RVA Yeast Labs

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces 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	Brettanomyces anomalus	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	Brettanomyces bruxellensis	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 Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	TYB184	This strain is the first single strain Brettanomyces isolate we are releasing all on its own because it deserves that distinct honor. Isolate TYB184 is literally the 184th isolate of yeast/bacteria that has made it through primary fermentation trials, was assigned an isolate number and carried into larger scale fermentation evaluation since our conception in 2013. This isolate is attenuative, produces a moderate acidic-like character and an ester profile of lemon/pineapple. Another notable characteristic of this isolate is the mild barnyard character it produces that doesn't take over the profile; rather, it balances the ester profile. The unique character balance in this strain is what makes it well suited for use on its own, in both primary and secondary fermentation. Approximately 15 billion cells/vial. Temperature: 72 - 85 ºF. Attenuation: 82%-88%. Flocculation: Medium-Low. Recommended by Nick for darker sours .	Isolated from a rustic farmhouse style beer produced in the Northeastern United States .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	TYB207	This isolate exhibits good attenuation, and produces a moderate acidic-like character and an ester profile the combination of which produces a character reminiscent of sweet tarts. It's a fruity, funky tartness that's refreshing and crisp. This strain is well-suited for primary and secondary fermentation. Approximately 15 billion cells/vial. Temperature: 70 - 82ºF , Attenuation: 80%-82%, Flocculation: Medium-Low.	Isolated from a Belgian-inspired brewery in the Northeastern United States .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	TYB261	This Brettanomyces isolate exhibits a mild tartness and soft funk with a solid backbone of tropical fruit esters (papaya, guava, pineapple, guinep). It's great primary fermenter, but really shines in secondary fermentation following up a primary fermentation that produces a lot of flavor compounds. This strain is a true flavor modulator, and the more raw material it has to work with the greater the complexity that will be achieved in the finished beer. Sequencing results revealed it's Brettanomyces bruxellensis. This strain will produce a massive, thick krausen, so be sure to use a blowoff tube or reserve plenty of fermentor headspace! Isolated from a unique mixed fermentation beer produced in the Midwestern United States .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	TYB307	This strain exhibits a lemony-tartness with hints of hay and mild barnyard funkiness, and has a crisp and dry finish. TYB307 is suitable for primary fermentation and extended aging. Fermentation temperature: 70-80ºF. Attenuation: 80-84%. Flucculation: low.	Isolated from a California brewery that utilizes an extensive and diverse array of organisms in the production of their wild/sour/funky beers .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	TYB415	This strain exhibits a strong profile of complex tropical fruit that is dominated by pineapple with a noticeable earthiness that adds a unique complexity and depth of character to the beer. TYB415 is suitable for primary fermentation and extended aging. Fermentation temperature: 70-80ºF. Attenuation: 82-86%. Flucculation: low.	Isolated from a brewer of all things sour and wild in the Mountain West .

White Labs

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

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	WLP650	Barnyard. Prominent Leathery aroma and flavor. Low levels of supporting tropical fruit in the aroma and flavor.	Not the same as WY's Brux. Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL .
Bruxellensis Trois Vrai	Dekkera bruxellensis	Brettanomyces bruxellensis	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 .	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. Might be the same as BSI Drie? Profile is very similar to BSI Drie . 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 . Bryan of Sui Generis blog and Devin Henry found that this culture contains two closely related B. bruxellensis strains with very different flavor profiles .
Anomalus	Dekkera anomala	Brettanomyces anomalus	WLP640	Typical barnyard funk with some fruitiness; claimed that it can be used for primary fermentation but a starter may be necessary.
Claussenii	Dekkera anomala	Brettanomyces anomalus	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" .	Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL . See also this MTF thread and this MTF thread which discuss the purity of this culture, and references Yakobson's data that indicates that it does not attenuate wort efficiently when purely isolated.
Lambicus	Dekkera bruxellensis	Brettanomyces bruxellensis	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 .	Different from WY's "lambicus". Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL .

Wyeast

Common Name	Species Name	Synonym Name	Lab/Package	Flavor/Aroma	Source Note
Anomalus	Dekkera anomala	Brettanomyces anomalus	Wyeast 5110		bottled stout - Burton on Trent, England. Discontinued in 2007 due to being misclassified . Cell count: 7.5 x 108 cells/mL .
Bruxellensis	Dekkera bruxellensis	Brettanomyces bruxellensis	Wyeast 5112	"sweaty horse blanket"	Not the same as WL's Brux. Cell count: 7.5 x 108 cells/mL .
Claussenii	Dekkera anomala	Brettanomyces anomalus	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 .
Lambicus	Dekkera bruxellensis	Brettanomyces bruxellensis	Wyeast 5526	Pie-cherry	Different from WL's "lambicus". Cell count: 7.5 x 108 cells/mL .

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.
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.
Escarpment Laboratories	Berliner Brett Blend	A blend of Berliner Brett 1 (Brettanomyces anomalus) and Berliner Brett 2 (Brettanomyces bruxellensis) for balanced, subtle Brett character in traditional Berliner Weisse and beyond. Flavour profile includes citrus, white wine, and peach. Secondary pitch rates only.
Escarpment Laboratories	MOTHERSHIP Brett Blend	This blend typically contains 10 individual strains. The character is highly dependent on fermentation conditions, but tends toward balanced, medium to high intensity Brett character.
Fermmento Labs (Brazil)	FB1 Tropical funky Bugs	Contains B. custersianus and 'B. anomalus.
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 .
Mainiacal Yeast	Hurricane Brett Blend	Mix of all 70 Brettanomyces strains at Mainiacal Yeast. Commercial brewery pitches always available, and occasionally available to homebrewers .
Propagate Lab	MIP-765 Native Brett Blend	Blend of Brettanomyces isolated from a number of natural sources. Isolates are from plums, natural wines, wild plants, and spontaneous fermentation. This blend will add complexity to any beer .
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 . 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 .
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 . 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 .
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 . 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 .
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 . 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 .
The Yeast Bay	Amalgamation II Brett Super Blend	Amalgamation II is a blend of 5 Brettanomyces isolates: Brettanomyces bruxellensis - Strain TYB184, Brettanomyces bruxellensis - Strain TYB207, Brettanomyces bruxellensis - Strain TYB261, and both Beersel Brettanomyces Blend isolates. The balanced funk of the Beersel isolates and TYB184, the sweet tart character of TYB207, and the tropical bouquet of the combined ester profile of lemon/pineapple/guava/mango/papaya contributed by all the isolates. Expect beers fermented with this blend to finish crisp, dry, tart and fruity with just a touch of funk on the finish. This blend produces noticeable character only 3-4 weeks into fermentation and is well suited for faster turnaround beers. Amalgamation II shines as a primary or secondary fermenter. 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 . Reportedly a fast fermenter

Using Brettanomyces

Primary versus Secondary Fermentation

Brettanomyces can be pitched into a beer at many points in the beer's fermentation life cycle. If used as the primary fermenter, the beer that is produced is often perceived as 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 on pitching rates for 100% Brettanomyces fermentation, as well as the fermentation characteristics of 100% Brettanomyces fermentation. If pitched into a beer that has already been fermented by Saccharomyces or if co-pitched with Saccharomyces, a wider range of flavors including the funkier flavors can be produced by changing some of the flavor compounds produced during the Saccharomyces fermentation into other flavor compounds (see the Brettanomyces Metabolism section above), although Brettanomyces can also produce funky phenols on its own (de novo) without phenolic precursors produced from Saccharomyces. A small cell count of Brettanomyces is plenty for creating these flavors if pitched after and co-pitched with Saccharomyces, and normally a starter is not necessary unless pitching Brettanomyces as the primary fermenter. See the Mixed Fermentation, Brettanomyces and Saccharomyces Co-fermentation, and Brettanomyces secondary fermentation experiment pages for more information on using Brettanomyces in secondary.

Starter Information

When pitching just Brettanomyces from a commercial pure or blended culture and no other microbes, it is recommended to make a starter for the culture. If the Brettanomyces is being pitched into secondary, no starter is necessary unless the brewer suspects that the Brettanomyces has lost a lot of viability due to age, heat exposure, etc., or prefers higher cell count pitches (current information suggests that there is no significant flavor difference between high and low pitching rates in secondary pitches of Brettanomyces; see Brettanomyces secondary fermentation experiment).

Starter wort made with dried malt extract at around 1.040 starting gravity is adequate for most strains of B. bruxellensis. For Brettanomyces strains such as many B. anomalus strains that don't ferment maltose, a mixture of 50% DME and 50% table sugar or dextrose at 1.040 starting gravity should be adequate. Yeast nutrient at the manufacturer's recommended rate can be added if sufficient growth is not observed .

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 .

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

Two Approaches to Starters

There are generally two approaches to handling Brettanomyces starters. The first is to use a stir plate set to a medium-high RPM with tin foil on top of the flask for 7-8 days, cold crash for a few days, and then decant the beer before pitching the sedimented yeast. The second approach is to use an orbital shaker set to 80 RPM to create a semi-aerobic environment (this means that the oxygen levels are low, but also not non-existent) for 7-8 days as described in The Brettanomyces project , 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 . 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 .

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 . 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 with foil covering the flask is 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, then they can opt 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 . 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. Even if the starter has a lot of acetic acid, the amount of acetic acid in the volume of a starter is fairly insignificant once diluted into a full batch of wort or beer. If the starter is not going to be used within a month, then an aerobic starter is not the best option since the presence of a lot of acetic acid will slowly kill the Brettanomyces over time. In this case, the starter should be lightly shaken (or occasionally manually stirred), and an airlock put in place on the flask in order to keep out most of the oxygen.

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

Maintaining a temperature of 77°-86°F/25°-30°C results in faster growth than lower temperatures and is recommended . Brettanomyces cell growth typically takes about 7-8 days to reach it's maximum growth , however some strains may grow at faster rates and finish in 3-4 days . When the starter turns a rich creamy color, it should be done within a few hours after this visual indication occurs . 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 . Since yeast calculators are based on S. cerevisiae or S. pastorianus cell density, using one of these tools for Brettanomyces starters will create an unexpectedly high cell count in reality. There is not currently enough data to accurately determine starter volumes for Brettanomyces, particularly because each strain and species have a different maximum cell density per mL of wort. However, pitching around 500-600 mL of a Brettanomyces starter for 5 gallons of 1.060 SG wort will achieve a pitching rate that is similar to lager yeast pitching rates, which has been recommended for 100% Brettanomyces Fermentation. Omega Yeast Labs is currently working on a project to create a more accurate Brettanomyces pitching rate calculator (it will also contain pitching rate calculations for specific strains of Saccharomyces, which is something that current yeast pitching calculators do not include) .

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

See also 100% Brettanomyces fermentation.

MYPG Growth Substrate and Other Laboratory Substrates

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 . Cells grown in MYPG also are better adapted to grow in wort . 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 .

For other suggested substrates for growing Brettanomyces and potentially other yeasts, see Laboratory Techniques.

Cell Counting

Bright-field and fluorescent images of B. clausenii, B. bruxellensis, and B. lambicus. The fluorescent images show the counting of the yeasts with and without declustering the bud and pseudohyphae. Source: https://link.springer.com/article/10.1007/s10295-016-1861-4. Used with permission from Brian Martyniak.

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 than methylene blue .

One of the problems with cell counting for Brettanomyces is that they tend to form pseudohyphae (elongated cells that are attached to one another) during growth. This makes counting the cells difficult. Martyniak et al. (2017) has proposed a new way of counting cells for Brettanomyces by using the fluorescence capability of a Nexcelom X2 image cytometer with stains created from acridine orange (AO) and propidium iodide (PI). The AO-PI stains only the nuclei of the individual cells, allowing them to be counted easily by the cytometer regardless of pseudohyphae formation. During their research, they found that a "clausenii" strain of B. anomalus formed more pseudohyphae than two strains of B. bruxellensis (one of which was refered to as a "lambicus" strain), and this corresponded with higher viability over time. It was therefore hyopthesized that pseudohyphae might play a role in the longer survival of some strains of Brettanomyces over others . It has been observed that pseudohyphae might be less common in Brettanomyces cultures that have been grown in wort versus lab media, making them easier to count , but this method makes it easy to count cells which are attached via pseudohyphae. This method has been criticized for its relatively high cost (both for the fluorescent microscope and the dyes) .

See also:

• MBAA Podcast interview with Leo Chan on automating cell counting with of Brettanomyces psuedohyphae.

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 .

Storing Brett

Major yeast labs will often store yeast in a -80°C laboratory freezer in a media/glycerol solution, although this option is generally not practical for brewers . The next best option for long-term storage of Brettanomyces is freezing with 10% glycerol in a home freezer. However, the effects of storing yeast at such a high and often variable temperature have not been evaluated scientifically. Traditionally Saccharomyces yeast has been stored on slants held in a refrigerator and can provide storage for a few months up to 2+ years, depending on the type of slant used (using mineral oil in slants has been shown to extend the life of stored Saccharomyces). Homebrewers, however, have reported poor survival of Brettanomyces on slants. Data from a MTF member showed promising results by buffering the slant media. In this data, Brettanomyces has stored well for up to 100 days on the buffered media. It is not known for how long viability will remain high on buffered slants. 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 effective solution and have been observed anecdotally to reduce the viability of Brettanomyces over a few months .

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 .

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 .

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 . 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 .

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

Fermentation Methods

• Mixed Fermentation
• Brettanomyces and Saccharomyces Co-fermentation
• 100% Brettanomyces Fermentation
• Spontaneous Fermentation

Tips From Brewers

The Yeast Bay

• Both Ed Coffey and Amos Browne have noted that the Lorchristi blend ages particularly well.

• 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 .

• MTF thread on The Yeast Bay Brett/Sacch flavor profiles.

Escarpment Laboratories

• "How to Choose a Brett Strain For Beer."

Pasteurization

Brettanomyces has complete thermal death at 122°F (50°C) for 5 minutes . See also Barrel Sanitizing and Pasteurization.

See Also

Additional Articles on MTF Wiki

• Brettanomyces Propagation Experiment
• Brettanomyces secondary fermentation experiment
• Brettanomyces Storage Survival Experiment
• 100% Brettanomyces Fermentation
• Crooked Stave Artisan Beer Project
• Scientific Publications
• Mixed Cultures
• Mixed Fermentation
• Brettanomyces and Saccharomyces Co-fermentation
• Debaryomyces
• Saccharomyces

External Resources

• Family tree of Brettanomyces, by Eureka Brewing Blog.
• Insight into the Brettanomyces Mitochondrial Genome, by Eureka Brewing Blog.
• National Collection of Yeast Cultures in the UK - Database on what compounds different species/strains can ferment.
• The Brettanomyces Project - Chad Yakobon's Brett research.
• The Mad Fermentationist - Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions
• The Mad Fermentationist - Comparison between English Ale yeast and Belgian Ale yeast primary fermentations, and Brett in secondary
• Blog Article on Brett and Glycosides by Cy Wood.
• Brettanomyces in Brewing the horse the goat and the barnyard, presentation by Chad Yakobson.
• Insight into the Dekkera anomala YV396 genome by Samuel Aeschlimann; self published on Eureka Brewing Blog.
• Esters - Table of esters and their smells.
• See the Phenol Explorer website for more information on sources of precursors.
• Scientific research on Brettanomyces by Mike Lentz et al., funded by the Oregon Wine Board.
• Old and new taxonomical classification of Brettanomyces and Dekkera.
• "Brettanomyces", Steve Piatz in Brew Your Own Magazine, October 2005.
• "100 year old Czech beer," by Dave Janssen (musings on the presence of Brettanomyces and high amounts of acetic and lactic acid in a 100 year old lager).
• "Brewing with Brettanomyces Yeast and Mixed Cultures" presentation by Chad Yakobson at BC Craft Brewers Guild, 2019.