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'''''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 <ref>[http://en.wikipedia.org/wiki/Brettanomyces Wikipedia. Brettanomyces. Retrieved 2/24/2015.]</ref><ref name="Avramova_2018">[https://www.nature.com/articles/s41598-018-22580-7 Brettanomyces bruxellensis population survey reveals a diploid-triploid complex structured according to substrate of isolation and geographical distribution. Marta Avramova, Alice Cibrario, Emilien Peltier, Monika Coton, Emmanuel Coton, Joseph Schacherer, Giuseppe Spano, Vittorio Capozzi, Giuseppe Blaiotta, Franck Salin, Marguerite Dols-Lafargue, Paul Grbin, Chris Curtin, Warren Albertin & Isabelle Masneuf-Pomarede. 2018. doi:10.1038/s41598-018-22580-7.]</ref>. Known for its barnyard, fecal, horsey, metallic or Band-Aid flavors, ''Brettanomyces'' continues to be unwelcome in many breweries and most wineries<ref>[https://www.cambridge.org/core/journals/journal-of-wine-economics/article/brettanomics-i-the-cost-of-brettanomyces-in-california-wine-production/295D206007B358EC1B07CEF4879BE06B Brettanomics I: The Cost of Brettanomyces in California Wine Production. Julian M. Alston, Torey Arvik, Jarrett Hart and James T. Lapsley. 2020. DOI: https://doi.org/10.1017/jwe.2020.20.]</ref>. 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#Commercial_Availability|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, around the same time that the first mammals emerged <ref name="Rozpędowska">[https://www.nature.com/articles/ncomms1305 Rozpędowska, E., Hellborg, L., Ishchuk, O. et al. Parallel evolution of the make–accumulate–consume strategy in Saccharomyces and Dekkera yeasts. Nat Commun 2, 302 (2011). https://doi.org/10.1038/ncomms1305.]</ref>. Both genera evolved independently to ferment sugar and produce ethanol <ref name="Schifferdecker">[http://onlinelibrary.wiley.com/doi/10.1002/yea.3023/pdf The wine and beer yeast Dekkera bruxellensis. Anna Judith Schifferdecker, Sofia Dashko, Olena P. Ishchuk, and Jure Piškur. 7 July 2014.]</ref><ref name="Gounot_2019">[https://www.biorxiv.org/content/10.1101/826990v1.full High complexity and degree of genetic variation in Brettanomyces bruxellensis population. Jean-Sébastien Gounot, Cécile Neuvéglise, Kelle C. Freel, Hugo Devillers, Jure Piškur, Anne Friedrich, Joseph Schacherer. 2019. DOI: https://doi.org/10.1101/826990 .]</ref>. Although first isolated from beer in 1889 by H. Seyffert of the Kalinkin Brewery in St. Petersberg and again in 1899 by scientists J. W. Tullo 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, he included these newly discovered yeasts in the genus ''Torula'' <refname="Gilliland_1961">[https://crescentcitybrewtalk.com/brettanomyces-i/ "BRETTANOMYCES I OCCURRENCE, CHARACTERISTICS, AND EFFECTS ON BEER FLAVOUR" by R. B. Gilliland, B.A., B.Sc, F.R.I.C. (Arthur Guinness Son & Co. (Dublin) Ltd., St. James’s Gate, Dublin). Received 21st Janurary, 1961.] See also [http://barclayperkins.blogspot.com/2013/06/when-was-brettanomyces-discovererd.html "When was Brettanomyces discovered?" Ron Pattenson. Shut Up About Barclay Perkins blog. 06/29/2013. retrieved Retrieved 08/18/2016.]</ref><ref>[http://breweryhistory.com/journal/archive/149/Yeast.pdf Ray Anderson. "ONE YEAST OR TWO? PURE YEAST AND TOP FERMENTATION". The Brewery History Society. 2012.]</ref>. 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'' <ref>[https://www.facebook.com/download/448702618652516/GB190328184A.pdf "Improvements in and connected with the Manufacture of English Beers or Malt Liquors and in the Production of Pure Yeast Cultures for use therein." Patent application by Hjelte Claussen for ''Brettanomyces''. A.D. 1903.]</ref>. Although Claussen saw the character from ''Brettanomyces'' as a desirable character in ales and , along with [[Hops#The_Freshening_Power_of_the_Hop_.28Hop_Creep.29|dry hop creep]], was identified its character as a quality the source of traditional English secondary fermentation during long aged ales, at some point ''Brettanomyces'' became identified as a contaminate in wineries contributing to their lasting high carbonation <ref>[https://archive.org/details/principlespracti00syke "The principles and breweries due to some practice of the phenolsbrewing" Sykes, acidsWalter John. London, and haze that it sometimes producesC. These phenols Griffin and acids have generally been described as "barnyard"Company, "burnt plastic"limited, "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 <ref name="smith_divol_2016">1907. Pgs 384-388.]</ref><ref name="Schifferdecker">[httphttps://onlinelibrarywww.wileyfacebook.com/doigroups/MilkTheFunk/10.1002permalink/yea4709953772366133 Gareth Young.3023/pdf Milk The wine Funk Facebook group thread about English brewers historically relying on Brettanomyces and beer yeast Dekkera bruxellensisdry hop creep for carbonation in long aged ales. Anna Judith Schifferdecker, Sofia Dashko, Olena P. Ishchuk, and Jure Piškur. 7 July 20106/17/2021.]</ref><ref name="Lucy_2015">[httphttps://www.ajevonlineyoutube.orgcom/contentwatch?v=9BwO7gbhdns Martyn Cornell interview on Craft Beer Channel, "The Time Is Now – reinventing the English IPA". 09/6628/3/379 Brettanomyces bruxellensis Aroma-Active Compounds Determined by SPME GC-MS Olfactory Analysis. C.M. Lucy Joseph, Elizabeth A. Albino, Susan E. Ebeler, Linda F. Bisson. 20152022.]</ref>. The general viewpoint of brewers (other than Lambic and Flanders red/brown brewers 8 minutes in Belgium) and vintners . Beer historian, [https://barclayperkins.blogspot.com/search?q=brettanomyces Ron Pattinson], has stated 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 was typically present in 1800''Brettanomyces'' beer s English aged beers such as pineapplestock ales, stone fruitspale ales, porters, and barrel aged IPA's that were shipped to some degree acetic acidIndia, have regained popularity with brewers outside and it was considered an important component of Belgium. Wine tasters have also described wines with certain both the flavor compounds derived profile of these beers and in protecting beer from contaminants via ''Brettanomyces'' as positive characteristics fermenting the majority of wineresidual sugars <ref>[https://www.crowdcast.io/e/IPA-Past-Present-Future/1 Ron Pattinson. It is important "History of IPA -1700s to keep in mind that individual tasters on tasting panels describe some flavor compounds as "negative" while others describe them as "positive2021" . Doug Piper's interview with Ron Pattinson. 07/25/2021.]</ref> (~56 and sometimes a mixed response is given by a taster 59 mins 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<ref>[https://www.facebook.com/milkthefunkthepodcast/videos/1097016944410369 Ron Pattinson. 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 negativelyMTF Live. Thus, lower intensity of some flavor compounds can be seen as more desirable02/10/2022. Overall, the enjoyment or displeasure of the various flavor compounds produced by ''Brettanomyces'' and at certain levels is completely subjective ]</ref name="Lucy_2015" />(45 minutes in).

It is common in scientific literature to see Following the names discovery of this yeast by Claussen, isolates of ''DekkeraBrettanomyces'' and were discovered in Belgian lambic beers in the 1920's. At this time, ''Brettanomyces'' used was proposed as the new genus name, with ''Dekkera'' being separating them from the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph teleomorph] version and genus ''BrettanomycesTorula'' being the [https:<ref name="Gilliland_1961" //en>.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph anamorph]. There are five The species within the genus of Brettanomyces: name 'bruxellensis'B. anomalus, meaning 'Brussels'in Latin, became the proposed species name for ''B. bruxellensis.''. This yeast species was then isolated from other industrial fermentations such as wine, cider, kombucha, kefir, olives, ''Band bioethanol production. Custers was the first to attempt to describe the rest of the genus using phenotypic characteristics in 1940. custersianus''In 1960, J. van der Walt observed some species of ''B. nanusBrettanomyces''formed ascospores, and this form of ''B. naardenensisBrettanomyces'' (one study on the genetics of was named ''B. nanus'' from 1990 classified Dekkera''B. nanus'' as belonging to another genus However, after the initial discovery of sporulating strains of yeast called ''EeniellaBrettanomyces'', however this behavior has not been agreed upon in more recent studies <ref>[http://onlinelibrary.wiley.com/doi/10.1002/yea.320060403/full Dekkerareported since, Brettanomyces and Eeniella: Electrophoretic comparison of enzymes and DNA–DNA homology. Maudy Th. Smith, M. Yamazaki, G. A. Poot. 1990.]</ref>). The species known as therefore some scientists prefer to use the term "''B. intermediusBrettanomyces'' and ''B" to refer to this genus. lambicus'' are considered synonyms Originally, a total of 9 species were attributed to the genus ''B. bruxellensisBrettanomyces'' <ref name="Agnolucci_2017">[https://link.springer.com/article/10.1007/s11274-017-2345-z Brettanomyces bruxellensis yeasts: impact on wine and winemaking. Monica Agnolucci, Antonio Tirellibut after gene technology was invented, Luca Cocolin, Annita Toffanin. 2017.]</ref>. Of some 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'' were changed (see [[Brettanomyces#Taxonomy|Taxonomy]] below) <ref name="smith_divol_2016Stenseels_2015_Essential"></ref><ref name="Schifferdecker"></ref><ref name="Steensels"></ref><ref>[httphttps://aemwww.asmacademia.org/content/80edu/1419646963/4398.full Assessing Genetic Diversity among Brettanomyces_Bruxellensis_Essential_Contributor_in_Spontaneous_Beer_Fermentations_Providing_Novel_Opportunities_for_the_Brewing_Industry Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome SequencingBruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry. Sam Crauwels, Bo Zhu, Jan Steensels, Pieter Busschaert, Gorik De Samblanx, Kathleen Marchald, Kris A. WillemsBrewingScience, Kevin JSept/Oct 2015 (Vol. Verstrepen and Bart Lieven68). 20142015.]</ref>. All of the other names such as the ones often used by yeast labs are derived by old nomenclature that is no longer used ([http://www.sciencedirect.com/science/article/pii/S0168160515001865#t0005 click here] for a table that lists old and new taxonomical nomenclature). Most ''Brettanomyces'' cultures from brewer's yeast labs are classified genetically as ''B. bruxellensis'' or ''B. anomalus''.

The morphology of Although Claussen and others saw the character from ''Brettanomyces'' can vary immensely from strain to strain (and species to species). Some strains can look similar as a desirable character in size English ales and shape to identified its character as a hallmark quality of traditional English ales, as pure cultures of ''S. cerevisiaeSaccharomyces'' under a microscopic imagewere introduced in English brewing in the early 20th century, while others are elongated or much smaller. This makes it difficult to identify ''Brettanomyces'' without DNA analysisbecame 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 <ref name="smith_divol_2016" /><ref name="Schifferdecker" /><ref name="Lucy_2015">[http://www.ajevonline.org/content/66/3/379 Brettanomyces bruxellensis Aroma-Active Compounds Determined by SPME GC-MS Olfactory Analysis. C.M. Lucy Joseph, Elizabeth A. Albino, Susan E. Ebeler, Linda F. Bisson. 2015.]</ref>. Morphologies The general viewpoint of brewers (other than Lambic brewers, Flanders red/brown brewers, and certain Trappist brewers in Belgium, as well as Berliner Weisse brewers in Berlin, Germany) and vintners became that ''Brettanomyces'' grown on agar plates can also be different from strain to strainis primarily a spoilage organism, and this still holds in most cases today. For exampleMore recently, however, Devin Henry found the positive flavor components that a sample of WLP648 that contained two closely related strains of have been identified in ''B. bruxellensisBrettanomyces'' grew completely differently on the same growth media. At firstbeer such as "pineapple", larger"stone fruits", slightly off-white colonies grew on the plates (this was the first strain)and to some degree acetic acid, and then a few days later the second strain grew as many smaller white-colored colonieshave regained popularity with brewers outside of Belgium. Other strains may appear Some winemakers and wine tasters have also described wines with certain flavor compounds derived from ''Brettanomyces'' as glossy or matted with jagged edges, etc. Morphology on agar plates can change depending on the type positive characteristics of growth media some wines <ref>[httphttps://brettanomycesprojectdaily.sevenfifty.com/dissertation/analysisthe-ofeverything-culturabilityguide-onto-various-media-agarbrettanomyces/ "The Everything Guide to Brettanomyces." GuildSomm. April 15, 2018. Retrieved 03/04/2021.]</ref><ref>[https://onlinelibrary.wiley.com/doi/full/morphological-traits10.1111/ Yakobsonnzg.12368 Siimes, ChadN. (2023). "Morphological Trains"Having a drink with awkward Brett: Brettanomyces, taste(s) and wine/markets. Masters DissertationNew Zealand Geographer, 1– 11. 2011https://doi. Retrieved 05org/1210.1111/2017nzg.12368.]</ref>. 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). This discrepancy in acceptability of flavor characteristics derived from ''Brettanomyces'' appears to be based on personal preference and experience. For example, in some cases and for some drinkers low levels of vinyl phenols produced by ''Brettanomyces'' contribute positively to wine, while higher amounts contribute negatively. Thus, a lower intensity of some flavor compounds can be seen as more desirable by some producers or consumers. Overall, the enjoyment or displeasure of the various flavor compounds produced by ''Brettanomyces'' and at certain levels is largely subjective <ref name="bryan_vraiLucy_2015" /><ref>[https://eurekabrewingwww.wordpressguildsomm.com/2012public_content/03features/articles/27b/brettanomyceskelli-bruxellensis-microscopy-pictureswhite/posts/ Samuel Aeschlimannbrettanomyces Brettanomyces: Science & Context. "Brettanomyces bruxellensis microscopy pictures"Kelli White. Eureka Brewing blogGuildSomm website. 03/1229/20122018. Retrieved 05/1210/20172019.]</ref>.

See also:* [https://www.facebook.com/groups/MilkTheFunk/permalink/3364694553558735/ A collection of early 1900's papers by Claussen and Alfred Chapman; MTF post by Cory Widmayer.] * [https://beerandbrewing-com.cdn.ampproject.org/c/s/beerandbrewing.com/amp/brasserie-de-la-sennes-yvan-de-baets-explains-saisons-greatest-myth-the Yvan de Baets believes that ''Brettanomyces'' probably played an important role in historical saison.]* [https://www.facebook.com/oddbreedwildales/videos/483806376609910 Presentation by Ron Pattinson on the role of ''Brettanomyces'' in historical English beer; hosted by Odd Breed Wild Ales on 02/15/2022.] ===Taxonomy=== It is common in scientific literature to see the names ''Dekkera'' and ''Brettanomyces'' used as the genus name, with ''Dekkera'' being the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph teleomorph] version and ''Brettanomyces'' being the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph 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 <ref>[http://onlinelibrary.wiley.com/doi/10.1002/yea.320060403/full Dekkera, Brettanomyces and Eeniella: Electrophoretic comparison of enzymes and DNA–DNA homology. Maudy Th. Smith, M. Yamazaki, G. A. Poot. 1990.]</ref><ref>[https://theyeasts.org/details/72/2050 The Yeasts website. "Brettanomyces nanus". Retrieved 11/05/2022.]</ref><ref>[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=13366&lvl=3&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep=1&srchmode=1&unlock NCBI Taxonomy Browser. "Brettanomyces". Retrieved 11/05/2022.]</ref>). The species previously known as ''B. intermedius'' and ''B. lambicus'' have recently been genetically analyzed and reclassified as strains of ''B. bruxellensis'' <ref name="Agnolucci_2017">[https://link.springer.com/article/10.1007/s11274-017-2345-z Brettanomyces bruxellensis yeasts: impact on wine and winemaking. Monica Agnolucci, Antonio Tirelli, Luca Cocolin, Annita Toffanin. 2017.]</ref>. 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'' <ref name="smith_divol_2016"></ref><ref name="Schifferdecker"></ref><ref name="Steensels"></ref><ref name="Crauwels_2014">[http://aem.asm.org/content/80/14/4398.full Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Sam Crauwels, Bo Zhu, Jan Steensels, Pieter Busschaert, Gorik De Samblanx, Kathleen Marchald, Kris A. Willems, Kevin J. Verstrepen and Bart Lieven. 2014.]</ref>. 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 ([http://www.sciencedirect.com/science/article/pii/S0168160515001865#t0005 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 <ref>[https://www.ncbi.nlm.nih.gov/pubmed/28160110 Brettanomyces acidodurans sp. nov., a new acetic acid producing yeast species from olive oil. Péter G, Dlauchy D, Tóbiás A, Fülöp L, Podgoršek M, Čadež N. 2017.]</ref>. 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 <ref name="Avramova_2018" />. 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'') <ref name="Gounot_2019" />. 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 <ref name="Avramova_2018" />. [https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637 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 [https://www.frontiersin.org/files/Articles/495404/fmicb-11-00637-HTML-r1/image_m/fmicb-11-00637-g002.jpg Figure 2 (family tree)] from the study <ref name="colomer_2020_genome">[https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637/full Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications. Marc Serra Colomer, Anna Chailyan, Ross T. Fennessy, Kim Friis Olsson, Lea Johnsen, Natalia Solodovnikova and Jochen Forster. 2020. DOI: https://doi.org/10.3389/fmicb.2020.00637.]</ref>. 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 <ref>[https://link.springer.com/article/10.1007/s00253-020-10750-5 Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system. Cristian Varela, Caroline Bartel, Cristobal Onetto, Anthony Borneman. Applied Microbiology and Biotechnology. 2020.]</ref>. ===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 [[Laboratory_Techniques#PCR.2FqPCR|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 <ref>[http://brettanomycesproject.com/dissertation/analysis-of-culturability-on-various-media-agar/morphological-traits/ Yakobson, Chad. "Morphological Trains". Masters Dissertation. 2011. Retrieved 05/12/2017.]</ref><ref name="bryan_vrai" /><ref>[https://eurekabrewing.wordpress.com/2012/03/27/brettanomyces-bruxellensis-microscopy-pictures/ Samuel Aeschlimann. "Brettanomyces bruxellensis microscopy pictures". Eureka Brewing blog. 03/12/2012. Retrieved 05/12/2017.]</ref>. While genetic (PCR) identification is required for any kind of confident identification of ''Brettanomyces'', specialized selective media can also help identify ''Brettanomyces''; see [[Laboratory_Techniques#Brettanomyces|Selective Media]].

''Brettanomyces'' has been thought to occur naturally on the skins of fruit such as apples and grapes. However, there are only a handful of reports of ''Brettanomyces'' being identified on the skins of fruit, and in some cases where ''Brettanomyces'' has been found, its abundance is extremely minimal <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.154 Lentz, M., Putzke, T., Hessler, R. and Luman, E. (2014), Genetic and physiological characterization of yeast isolated from ripe fruit and analysis of fermentation and brewing potential, J. Inst. Brew., 120: 559– 564. DOI: 10.1002/jib.154.]</ref><ref name="Comitini">[https://www.frontiersin.org/articles/10.3389/fmicb.2019.00415/abstract Occurrence of Brettanomyces bruxellensis on grape berries and in related winemaking cellar. Francesca Comitini, Lucia Oro, Laura Canonico, Valentina Marinelli, Maurizio Ciani. 2019. DOI: 10.3389/fmicb.2019.00415.]</ref><ref name="Renouf_2007">[https://www.sciencedirect.com/science/article/pii/S0944501306000231?via%3Dihub Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Vincent Renouf, Aline Lonvaud-Funel. 2007. DOI: https://doi.org/10.1016/j.micres.2006.02.006.]</ref>. In contrast, there are also studies that indicate ''Brettanomyces'' only being found during or after food processing, which indicates that the processing equipment may be the primary source for the ''Brettanomyces''. In addition, ''Brettanomyces'' has been isolated in abundance from the surfaces of equipment/processed materials in wineries and breweries <ref name="smith_divol_2016" /><ref name="Schifferdecker" /><ref name="Loureiro_2003">[https://www.ncbi.nlm.nih.gov/pubmed/12892920 Spoilage yeasts in the wine industry. Loureiro V, Malfeito-Ferreira M. 2003.]</ref><ref name="Steensels" /><ref name="Barata_2008">[https://www.ncbi.nlm.nih.gov/pubmed/18077036 Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. f Barata A, Caldeira J, Botelheiro R, Pagliara D, Malfeito-Ferreira M, Loureiro V. 2008.]</ref> (Table 1). For example, an ongoing survey of wild yeasts in different regions of United States wilderness areas which isolated nearly 2,000 isolates with 262 unique species has not yet found a single occurrence of ''Brettanomyces'' in the wild (so far they have only surveyed non-human inhabited wild areas of the US and Alaska; substrates sampled included leaves, soil, bark, moss, mushrooms, needles, pine cones, twigs/wood, and other plant matter) <ref>[https://www.biorxiv.org/content/10.1101/2021.07.13.452236v1 Substrate, temperature, and geographical patterns among nearly 2,000 natural yeast isolates. William J. Spurley, Kaitlin J. Fisher, Quinn K. Langdon, Kelly V. Buh, Martin Jarzyna, Max A. B. Haase, Kayla Sylvester, Ryan V. Moriarty, Daniel Rodriguez, Angela Sheddan, Sarah Wright, Lisa Sorlie, Amanda Beth Hulfachor, Dana A. Opulente, Chris Todd Hittinger. bioRxiv 2021.07.13.452236; doi: https://doi.org/10.1101/2021.07.13.452236.]</ref>. It is therefore unclear that ''Brettanomyces'' found on grape skins originated there or from the industrial processing where it is more abundant. It is also thought to disperse via fruit-flies (called "vectors" in the scientific literature), similar to how ''Saccharomyces'' travels, although direct evidence for this has been reported rarely and only on fruit-flies in wineries that are likely to come into contact with equipment/processed material that is already contaminated with ''Brettanomyces'' <ref>[https://youtu.be/G2nhUM5PIrg?t=309 Dr. Bryan Heit. BotB - Where (Do) The Wild Brettanomyces Roam?. ~5 mins in. Retrieved 07/10/2022.]</ref><ref name="Renouf_2007" /><ref name="Steensels">[http://www.sciencedirect.com/science/article/pii/S0168160515001865 Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.]</ref><ref name="Barata_2008" /><ref name="Loureiro_2003" />. ''Brettanomyces'' is known to be difficult to grow in a lab due to slow growth, specific nutrient requirements, or perhaps because of a "VBNC" state (see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild ''Brettanomyces'']] for more information), which may account for the lack of evidence for fruit being the primary natural habitat for ''Brettanomyces''. More recently, techniques have been invented to more easily isolate and grow ''Brettanomyces'' <ref name="Renouf_2007" /><ref name="Comitini" />. There is also significant evidence that the natural habitat of ''Brettanomyces'' might actually be the root systems of certain plants, known as the [https://www.nature.com/scitable/knowledge/library/the-rhizosphere-roots-soil-and-67500617/ "rhizosphere"]. The rhizosphere refers to the complex symbiotic community of microbe populations that live on and around the root system of plants. Wild strains of ''Brettanomyces'' have been found in the root systems of dill, common beans, sunflowers, maize, corn, jute, cassava, and grey mangroves found in the estuaries of Indonesia <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1111/aab.12309 Weisany, W., Raei, Y., Salmasi, S., Sohrabi, Y. and Ghassemi-Golezani, K. (2016), Arbuscular mycorrhizal fungi induced changes in rhizosphere, essential oil and mineral nutrients uptake in dill/common bean intercropping system. Ann Appl Biol, 169: 384-397. https://doi.org/10.1111/aab.12309.]</ref><ref>[https://archive.aessweb.com/index.php/5003/article/view/3333 I.O, S. ., & G.P, O. . (2012). Diversity of Fungal Populations in Soils Cultivated With Cassava Cultivar TMS 98/0505. Journal of Asian Scientific Research, 2(3), 116–123. Retrieved from https://archive.aessweb.com/index.php/5003/article/view/3333.]</ref><ref>[https://www.ajol.info/index.php/swj/article/view/149513 Rhizosphere and non-rhizosphere soil mycoflora of Corchorus olitorius (Jute). G.S. Olahan, I.O. Sule, T Garuba, Y.A. Salawu. Science World Journal. 2016.]</ref><ref>[https://ojs.unud.ac.id/index.php/jbb/article/view/36023 NOERFITRYANI, Noerfitryani; HAMZAH, Hamzah. THE EXISTENCE OF ENTOMOPATHOGENIC FUNGI ON RICE PLANTS RHIZOSPHERE. International Journal of Biosciences and Biotechnology, p. 12-24, dec. 2017. ISSN 2655-9994. doi: https://doi.org/10.24843/IJBB.2017.v05.i01.p02.]</ref><ref>[https://www.sciencedirect.com/science/article/abs/pii/S2452219818300259 Marcela Sarabia, Saila Cazares, Antonio González-Rodríguez, Francisco Mora, Yazmín Carreón-Abud, John Larsen, Plant growth promotion traits of rhizosphere yeasts and their response to soil characteristics and crop cycle in maize agroecosystems, Rhizosphere, Volume 6, 2018, Pages 67-73, ISSN 2452-2198, https://doi.org/10.1016/j.rhisph.2018.04.002.]</ref><ref>[https://www.sciencedirect.com/science/article/abs/pii/S1049964419303238 Nivien A. Nafady, Mohamed Hashem, Elhagag A. Hassan, Hoda A.M. Ahmed, Saad A. Alamri. The combined effect of arbuscular mycorrhizae and plant-growth-promoting yeast improves sunflower defense against Macrophomina phaseolina diseases. Biological Control. Volume 138, 2019, 104049. ISSN 1049-9644, https://doi.org/10.1016/j.biocontrol.2019.104049.]</ref><ref>[http://ejurnal.its.ac.id/index.php/sains_seni/article/view/5613 Isolation and Characterization of Yeast from Rhizosphere Avicennia Marina Wonorejo. Sitatun Zunaidah, Nur Hidayatul Alami. 2014. DOI: 10.12962/j23373520.v3i1.5613.]</ref>. See Dr. Bryan Heit's video [https://www.youtube.com/watch?v=G2nhUM5PIrg "Where (Do) The Wild Brettanomyces Roam?"] and [https://www.facebook.com/groups/MilkTheFunk/posts/5940213029340195 his comments in Milk The Funk], as well as [https://www.youtube.com/watch?v=BrR7G_YyfmA "Philip Poole. Plant Control of the Rhizosphere Microbiome"]. For documented isolation attempts from plant rhizospheres, see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild Yeast Isolation]]. The occurrence of ''Brettanomyces'' has been more commonly identified in industrial food processing areas (wine, beer, kombucha, soft drinks, dairy products, tea, sourdough, etc.) <ref name="Crauwels_2016">[https://academic.oup.com/femsyr/article-abstract/17/1/fow105/2670560/Fermentation-assays-reveal-differences-in-sugar?redirectedFrom=fulltext Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains. Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains. Sam Crauwels, Filip Van Opstaele, Barbara Jaskula-Goiris, Jan Steensels, Christel Verreth, Lien Bosmans, Caroline Paulussen, Beatriz Herrera-Malaver, Ronnie de Jonge, Jessika De Clippeleer, Kathleen Marchal, Gorik De Samblanx, Kris A. Willems, Kevin J. Verstrepen, Guido Aerts, and Bart Lievens. 2016]</ref>. 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 <ref name="Tiukova_2019">[https://www.mdpi.com/2076-2607/7/11/489 Assembly and Analysis of the Genome Sequence of the Yeast Brettanomyces naardenensis CBS 7540. Ievgeniia A. Tiukova, Huifeng Jiang, Jacques Dainat, Marc P. Hoeppner, Henrik Lantz, Jure Piskur, Mats Sandgren, Jens Nielsen, Zhenglong Gu, and Volkmar Passoth. 2019. DOI: https://doi.org/10.3390/microorganisms7110489.]</ref>. ''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). This 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) <ref>[http://www.sciencedirect.com/science/article/pii/S0956713513002284 Screening of yeast mycoflora in winery air samples and their risk of wine contamination. E. Ocón, P. Garijo, S. Sanz, C. Olarte, R. López, P. Santamaría, A.R. Gutiérrez. Food Control Volume 34, Issue 2, December 2013, Pages 261–267.]</ref>. 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'' (although evidence for this is lacking).  ''Brettanomyces'' is commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries(although the median occurrence of ''Brettanomyces'' in barrels may be very low to none within a given winery or brewery depending on their hygiene and other factors <ref>[https://link.springer.com/article/10.1007/s00217-011-1523-8 Guzzon, R., Widmann, G., Malacarne, M. et al. Survey of the yeast population inside wine barrels and the effects of certain techniques in preventing microbiological spoilage. Eur Food Res Technol 233, 285–291 (2011). https://doi.org/10.1007/s00217-011-1523-8.]</ref><ref>[https://agris.fao.org/agris-search/search.do?recordID=IT2007601151 Fontanot, S.; Ninino, M.E.; Comi, G.; Elimination of Dekkera/Brettanomyces from barriques of the Italian CDO Isonzo area. Controlled Designation of Origin; Friuli-Venezia Giulia. 2006.]</ref>). 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 <ref name="Agnolucci_2017" /><ref name="Cartwright_2018">[http://www.ajevonline.org/content/early/2018/05/23/ajev.2018.18024 Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam. Zachary M. Cartwright, Dean A. Glawe, Charles G. Edwards. 2018. DOI: 10.5344/ajev.2018.18024.]</ref>, 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 <ref name="Cartwright_2018" />. 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#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment. Fabrizio, Vigentini, Parisi,Picozzi, Compagno, Foschino. 2015.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1466856417310068 Control of Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and gaseous ozone. Francesco Craveroa, Vasileios Englezos, Kalliopi Rantsiou, Fabrizio Torchio, Simone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. 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 [[Quality_Assurance#Biofilms|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'' <ref name="smith_divol_2016">[http://www.sciencedirect.com/science/article/pii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Brendan D. Smith, Benoit Divol. June 2016.]</ref>.

Unlike most genres 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 <ref name="Crauwels1" /><ref name="Agnolucci_2017" />), a pH as low as 2 <ref>[http://www.winesandvines.com/template.cfm?section=news&content=141954 Wines and Vines. New Research on Role of Yeast in Winemaking; report on a presentation by David Mills and Lucy Joseph from UC Davis. 11/14/2014. Retrieved 08/16/2015.]</ref>, and environments with low nitrogen <ref name="Schifferdecker"></ref> and low sugar sources <ref name="Smith_2018">[https://www.sciencedirect.com/science/article/pii/S0740002017308249 The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium. Brendan D.Smith and Benoit Divol. 2018. DOI: https://doi.org/10.1016/j.fm.2017.12.011.]</ref>. It has been reported that ''B. bruxellensis'' is more tolerant of high levels of bicarbonate than compared to ''S. cerevisiae'' (levels above 100 mg/l slow the fermentation of ''B. bruxellensis'', but do not completely inhibit it, with up to 400 mg/l being tested in one study) <ref name="Thompson-Witrick_2022">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2021.1940654 Katherine A. Thompson-Witrick & Eric R. Pitts (2022) Bicarbonate Inhibition and Its Impact on Brettanomyces bruxellensis Ability to Produce Flavor Compounds, Journal of the American Society of Brewing Chemists, 80:3, 270-278, DOI: 10.1080/03610470.2021.1940654.]</ref>. 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 <ref name="Smith_2017">[https://www.sciencedirect.com/science/article/pii/S0740002017308249 The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium. Brendan D. Smith, Benoit Divol. 2017.]</ref>. Some strains are able to utilize ethanol, glycerol, acetic acid, and malic acid when no other sugar sources are available <ref name="Smith_2018" />. 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 <ref name="smith_divol_2016"></ref>. [https://www.winesandvines.com/news/article/200000/New-Tools-to-Limit-Wine-Spoilage 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 [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|fermentation temperature]] for more information <ref>[http://www.ajevonline.org/content/early/2017/01/05/ajev.2017.16102 Interactions between Storage Temperature and Ethanol that Affect Growth of Brettanomyces bruxellensis in Merlot Wine. Taylor A. Oswald, Charles G. Edwards. 2017.]</ref>. ''Brettanomyces'' is also tolerant of IBU's, and there is some evidence that ''Brettanomyces'' is only inhibited by very high IBU's. One study reported that one strain of ''B. bruxellensis'' was inhibited by exposure to 250 mg/L of isomerized hop extract (roughly 250 IBU). Very little inhibition occurred at 150 IBU and about a third of the cells were inhibited at 200 IBU. The inhibited cells were recoverable in YPD media treated with catalase enzyme. In comparison, ''S. cerevisiae'' can be inhibited by 500 mg/L of iso-alpha acids <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2022.902110/full "Transcriptome Analysis of Viable but Non-Culturable Brettanomyces bruxellensis Induced by Hop Bitter Acids". He Yang, Zhao Junfeng, Yin Hua, Deng Yuan. Frontiers in Microbiology. 2022. DOI: 10.3389/fmicb.2022.902110 .] See also [https://www.facebook.com/groups/MilkTheFunk/posts/7473091549385661/ this MTF post]</ref>.

The genetic diversity of ''Brettanomyces'' is known for not producing much glycerol in beerparticularly wide. [https://en.wikipedia.org/wiki/Glycerol Glycerol] is a colorless, sweet-tastingFor example, viscous liquid one study that is thought to be an important contributor to analyzed the mouthfeel whole genomes of beer53 strains of ''B. Glycerol is produced as a stress response by a wide range bruxellensis'' found that the overall genetic diversity between different strains of microbes, including ''SB. cerevisiaebruxellensis'', and various species and was higher than strains of ''DebaryomycesS. cerevisiae''(however, the entire gene set, known as the ''Candidapangenome'', of all the genes among all of the strains of ''LachanceaB. bruxellensis'', and is much smaller than the entire gene set of ''ZygosaccharomycesS. cerevisiae'') <ref name="Gounot_2019" />. Despite not producing amounts of glycerol Some studies have indicated that are perceivable in beer, some strains of ''Brettanomyces B. bruxellensis'' actually produce glycerol which is stored inside of their cells as a response have adapted to osmotic stressspecific environments. They can also uptake glycerol into their cellsFor example, one study found that strains of ''B. Doing so allows bruxellensis'' isolated from wine had 20 genes involved in the cells to survive osmotic pressure <ref>[https://wwwmetabolism of carbon and nitrogen, whereas strains isolated from beer did not.sciencedirect This indicated that ''B.com/science/article/pii/S0740002013001251?via%3Dihub Osmotic stress response bruxellensis'' strains living in wine have adapted to the harsher environment of wine yeast Dekkera bruxellensis. Silvia Galafassi, Marco Toscano, Ileana Vigentin, Jure Piškur, Concetta Compagno. 2013.]</refname="smith_divol_2016"></ref>[https://academic.oup.com/femsle/advance-article-abstract/doi/10.1093/femsle/fny020/4828327?redirectedFrom=fulltext Osmotolerance Another study found that one out of Dekkera bruxellensis 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 role of two Stl glycerol-proton symportersbeer/soda strains did not. Jana ZemančíkováThe wine strains were also more resistant to sulfites, Michala Dušková, Hana Elicharová, Klára Papoušková, Hana Sychrová. 2018.]which are commonly used in the wine industry to prevent microbial contamination <ref name="Crauwels_2016" /ref>. It is currently not known how many strains are capable The whole genome sequencing of producing glycerol internally, or if this amount one strain of glycerol has any impact on perceived mouthfeel of a beer if a substantial amount ''B. naardenensis'' and lambic strains of ''BrettanomycesB. bruxellensis'' cells eventually autolyze (see [https://www.facebook.com/groups/MilkTheFunk/permalink/2003626776332193/ this MTF thread]). The role found that they are missing the genes associated with nitrate utilization, indicating that the assimilation of glycerol in creating mouthfeel nitrates is debatable not required to survive in beer, perhaps because of the wine world abundance of nitrogen from other sources found in beer <ref>[https://www.winesandvines.com/features/article/68760 Tim Patterson. name="Many Roads to MouthfeelTiukova_2019". Wines & Vines Magazine. Nov 2009. Retrieved 03/23/2018.]><ref name="colomer_2020_genome" /ref>.

The genetic diversity addition of vitamins can have a positive impact on ''Brettanomyces'' is particularly widegrowth. 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 <ref>[http://onlinelibrary.wiley.com/doi/10.1002/jib.385/full The influence of thiamine and riboflavin on various spoilage microorganisms commonly found in beer. Barry Hucker, Melinda Christophersen, Frank Vriesekoop. 2017.]</ref>. Some studies have indicated that 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 ''BBrettanomyces'' to grow. bruxellensis The presence of alcohol can increase the dependence on vitamins for some strains of ''Brettanomyces'' have adapted to specific environmentsgrow. For example, one study found that Myo-inositol (vitamin B8) and thiamine (vitamin B1) were required by two strains of ''B. bruxellensis'' isolated from wine had 20 genes involved when grown in the metabolism of carbon and nitrogen, whereas strains isolated from beer did 10% ethanol but notin 0% ethanol. This indicated 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'' strains living in wine have adapted to the harsher environment of wine <ref name="smith_divol_2016"></ref>. Another study found Other studies contradict these previously mentioned findings, showing that one out of thiamine was not required by the two strains of ''B. bruxellensis'' tested , that were isolated from soda could not ferment maltosepyridoxine was required, and only biotin was not required. These discrepancies between scientific studies are probably due to the genetic differences between the strains isolated from wine were able to grow in wine selected, the growth media chosen by the scientists, and /or the beergrowth conditions <ref>[http://soda strains did notwww.mdpi. The wine strains were also more resistant com/2311-5637/2/3/17 Use of Nutritional Requirements for Brettanomyces bruxellensis to sulfites, which are commonly used Limit Infections in the wine industry to prevent microbial contamination Wine. Nicolas H. von Cosmos and Charles G. Edwards. 2016.]</ref name="Crauwels_2016" />.

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 5 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. 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. 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 [https://link.springer.com/article/10.1007%2FBF02539015 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) <ref name="Avramova_2018" />. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/2022801681081369/ Richard Preiss's discussion of this study on MTF]. ===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 <ref name="smith_divol_2016"></ref>) <ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4549.2012.00702.x/abstract Removal of Brettanomyces Bruxellensis from Red Wine Using Membrane Filtration. Umiker, Descenzo, Lee, and Edwards. 04/24/2012.]</ref>. 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 <ref name="Agnolucci_2017" />. This dosage of molecular SO₂ requires a total amount of SO₂ that is beyond legal limits (350 mg/l <ref>[https://grapesandwine.cals.cornell.edu/sites/grapesandwine.cals.cornell.edu/files/shared/documents/Research-Focus-2011-3.pdf Sulphur Dioxide Content of Wines: the Role of Winemaking and Carbonyl Compounds. Nick Jackowetz, Erhu Li, and Ramón Mira de Orduña. 2011.]</ref>; see this [https://grapesandwine.cals.cornell.edu/newsletters/appellation-cornell/2012-newsletters/issue-12/article-contains-sulfites/ 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 is thought has been demonstrated that the wine strains have adapted to the conditions of winemakers adding SO₂ to wine <ref name="Bartel_2021">[https://academic.oup.com/femsyr/advance-article-abstract/doi/10.1093/femsyr/foab036/6293842 Caroline Bartel, Michael Roach, Cristobal Onetto, Chris Curtin, Cristian Varela, Anthony Borneman, Adaptive evolution of sulfite tolerance in Brettanomyces bruxellensis, FEMS Yeast Research, 2021;, foab036, https://doi.org/10.1093/femsyr/foab036.]</ref><ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2018.01260/full Molecular Diagnosis of Brettanomyces bruxellensis’ Sulfur Dioxide Sensitivity Through Genotype Specific Method. Avramova M, Vallet-Courbin A, Maupeu J, Masneuf-Pomarède I, Albertin W. 2018. DOI: 10.3389/fmicb.2018.01260.]</ref>. There is also variability between wine strains in their ability to tolerate SO₂ <ref>[https://www.sciencedirect.com/science/article/abs/pii/S2212429221000250 Jessica Lleixà, Maria Martínez-Safont, Isabelle Masneuf-Pomarede, Maura Magani, Warren Albertin, Albert Mas, Maria C. Portillo. Genetic and phenotypic diversity of Brettanomyces bruxellensis isolates from ageing wines. Food Bioscience. Volume 40. 2021. ISSN 2212-4292. https://doi.org/10.1016/j.fbio.2021.100900.]</ref>. For example, ''Brettanomyces'' strains isolated from sweet wine tend to be more tolerant of sulfur dioxide than strains isolated from dry wine <ref>[https://www.sciencedirect.com/science/article/pii/S0740002018303988 Sulfur dioxide response of Brettanomyces bruxellensis strains isolated from Greek wine. Maria Dimopoulou, Magdalini Hatzikamari, Isabelle Masneuf-Pomarede, Warren Albertin. 2018. DOI: https://doi.org/10.1016/j.fm.2018.10.013.]</ref>. 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 [[Quality_Assurance#VBNC_In_Yeast|VBNC in Yeast]])<ref>[https://www.sciencedirect.com/science/article/abs/pii/S0740002020302069 Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Adriana Nunes de Lima, Rui Magalhães, Francisco Manuel Campos, José António Couto. 2020. DOI: https://doi.org/10.1016/j.fm.2020.103617.]</ref>. 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 [[Saccharomyces#Killer_Wine_Yeast|Killer Wine Yeast]] for more information). [http://www.laboratoriosenosan.com/en/effectiveness-of-kaolin-silver-complex/ 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 <ref>[https://www.ncbi.nlm.nih.gov/pubmed/29666535?dopt=Abstract Effect of kaolin silver complex on the control of populations of Brettanomyces and acetic acid bacteria in wine. Izquierdo-Cañas PM, López-Martín R, García-Romero E, González-Arenzana L, Mínguez-Sanz S, Chatonnet P, Palacios-García A, Puig-Pujol A. 2018. DOI: 10.1007/s13197-018-3097-y.]</ref>. Other proposed replacements for SO₂ as a way to inhibit ''Brettanomyces'' in wine include [https://en.wikipedia.org/wiki/Pascalization high pressure processing] and [https://www.sciencedirect.com/science/article/abs/pii/S0255270106001929 pulsed electric fields] <ref>[https://www.sciencedirect.com/science/article/pii/S1466856418302972 SO2, high pressure processing and pulsed electric field treatments of red wine: Effect on sensory, Brettanomyces inactivation and other quality parameters during one year storage. Sanelle Van Wyk, Mohammed M. Farid, Filipa V.M. Silva. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.06.016.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1466856418307409 Pulsed electric field treatment of red wine: Inactivation of Brettanomyces and potential hazard caused by metal ion dissolution. Sanellevan Wyk, Filipa V.M. Silva, Mohammed M.Farid. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.11.001.]</ref>.

The addition A study by Cibrario et al. (2019) looked at the genome of ''B. bruxellensis'' strains in bottles of vitamins 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 a positive impact on 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 ''BrettanomycesB. bruxellensis'' growthstrains become long-term residents in wineries. For exampleSome 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 ''BrettanomycesB. bruxellensis''becomes rather sedentary and a constant resident in wineries, it can also adapt to the different winemaking conditions in different regions once it' does not need riboflavin s been transported (vitamin B2different grapes, different climates, different fermentation temperatures, etc.) or thiamine (vitamin B1) in order , including adapting to growimproved modern hygienic practices such as higher SO₂ treatment. Overall, the presence of either or both of these two vitamins encourages results from this study suggest that ''Brettanomyces'' growth is able to adapt to living alongside certain human industries and has done so for at least a couple of centuries <refname="Cibrario_2019">[httphttps://onlinelibrarywww.wileybiorxiv.comorg/doicontent/10.10021101/jib.385/full The influence of thiamine 763441v1 Brettanomyces bruxellensis wine isolates show high geographical dispersal and riboflavin on various spoilage microorganisms commonly found long remanence in beercellars. Alice Cibrario, Marta Avramova, Maria Dimopoulou, Maura Magani, Cécile Miot-Sertier, Albert Mas, Maria C. Portillo, Patricia Ballestra, Barry HuckerView ORCID ProfileWarren Albertin, Melinda ChristophersenIsabelle Masneuf-Pomarede, Frank VriesekoopMarguerite Dols-Lafargue. 20172019. DOI: https://doi.org/10.1101/763441.]</ref>. Other vitamins such as p-aminobenzoic acid The genetic differences between the fermentation substrates (PABA)beer, folic acid (vitamin B9)wine, nicotinic acid (vitamin B3etc.)were lower but still significant, pantothenic acid (vitamin B5) are also not required and this was explained by the frequent cross-over of equipment such as wine barrels being used for most strains of ''Brettanomyces'' to growbeer fermentation. The presence When comparing the geographic differences, they found geography contributed only 5% of genetic differences, while geography explained more than 50% of alcohol can increase 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 dependence on vitamins for some wine strains of ''Brettanomyces'' to growtravel across the globe. For example, MyoThey also found that although one study reported spore-inositol (vitamin B8) and thiamine (vitamin B1) were required by two strains forming versions of ''B. bruxellensis'' when grown in 10% ethanol but not in 0% ethanol. Biotin (vitamin B7referred to as ''Dekkera bruxellensis'') is the one exception, and it was found that the lack genetic makeup of biotin inhibited the growth of some analyzed strains determined their ability to sporulate to be non-existent or rare (only one study that we know of by [https://link.springer.com/article/10.1007%2FBF02539015 Walt and Kerken in 1960] has reported sporulation in ''B. bruxellensisBrettanomyces''. Other studies contradict these previously mentioned findingsonly on specific agar types with vitamins added, showing indicating that thiamine was not required by the strains of sporulation in ''B. bruxellensisBrettanomyces'' 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 is extremely rare) <refname="Avramova_2018" />. See also [httphttps://www.mdpifacebook.com/2311-5637groups/MilkTheFunk/2permalink/32022801681081369/17 Use Richard Preiss's discussion of Nutritional Requirements for Brettanomyces bruxellensis to Limit Infections in Winethis study on MTF]. Nicolas HA study by [https://academic. von Cosmos and Charles Goup. Edwardscom/femsyr/advance-article-abstract/doi/10. 20161093/femsyr/foab036/6293842 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 <refname="Bartel_2021" />.

[[File:Custers Headstone.jpg|thumb|300px|right|Dr. Custers memorial. Photo by [https://www.facebook.com/groups/MilkTheFunk/permalink/3061430667218460/ Jan Beekaa Lemmens].]] Like ''Saccharomyces'', ''Brettanomyces'' is [https://en.wikipedia.org/wiki/Crabtree_effect Crabtree] positive (produces alcohol in the presence of oxygen and high sugar concentration), and is [https://en.wikipedia.org/wiki/Petite_mutation petite] positive (unable to grow without carbon sources, and forms small colonies when able to grow on growth media) <ref name="smith_divol_2016"></ref>. 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 "[https://en.wikipedia.org/wiki/Pasteur_effect 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 <ref name="Agnolucci_2017" />. This was initially dubbed the "negative Pasteur effect" by Custers, and later the "Custers effect" by W. A. Scheffers <ref name="yakobson_introduction"></ref><ref>[http://link.springer.com/article/10.1007/BF02157944 On the inhibition of alcoholic fermentation in Brettanomyces yeasts under anaerobic conditions. W. A. Scheffers. 1961.]</ref>. A notable exception to this is the species ''B. naardenensis'', which only produces ethanol when oxygen is limited <ref name="Tiukova_2019" />.  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 <ref>[http://www.sciencedirect.com/science/article/pii/S0308814606002457 The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: A review. R.Suárez, J.A.Suárez-Lepe, A.Morata, F.Calderón. 2007.]</ref><ref>[https://search.proquest.com/docview/733013604?pq-origsite=gscholar Fermentation characteristics of Dekkera bruxellensis strains. Blomqvist, Johanna; Eberhard, Thomas; Schnürer, Johan; Passoth, Volkmar. 2010.]</ref><ref>[https://books.google.com/books?id=XlHuCAAAQBAJ&pg=PA436&lpg=PA436&dq=oxidative+yeast&source=bl&ots=poULkx-VUd&sig=eqRoMnh8vIfC0NvBCqvbl6ghrSA&hl=en&sa=X&ved=0ahUKEwiGhZOXmdPXAhWHg1QKHdf5A6s4FBDoAQgnMAA#v=onepage&q=oxidative%20yeast&f=false Wofl, Laus. "Nonconventional Yeasts in Biotechnology: A Handbook." Springer Science & Business Media, Dec 6, 2012. Pg 436.]</ref><ref>[http://www.wyeastlab.com/wild-beer-brewing "Wild Beer Brewing" Wyeast website. Retrieved 11/22/2017.]</ref><ref>[http://laboratoryresearch.blogspot.com/2008/07/yeasts-and-yeastlike-fungi.html?m=1 "YEASTS AND YEASTLIKE FUNGI" Do You Know? blog. 2008. Retrieved 11/22/2017.]</ref>.

''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'' <ref name="roach_2019">[https://www.biorxiv.org/content/10.1101/805721v2 New genome assemblies reveal patterns of domestication and adaptation across Brettanomyces (Dekkera) species. Michael J. Roach, Anthony R. Borneman. 2019. DOI: https://doi.org/10.1101/805721.]</ref>. 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'' <ref name="Steensels"></ref><ref name="smith_divol_2016"></ref><ref name="Smith_2018" />. For example, the species ''B. naardenensis'' can ferment a wide range of carbon sources, including galactose, maltose, xylose, trehalose, cellobiose, rhamnose, and arabinose <ref name="Tiukova_2019" />. Acetic acid, glycerol, succinic acid, and ethanol are only consumed if oxygen is present <ref name="smith_divol_2016"></ref>. The addition of small amounts of O2 stimulates glucose fermentation, as well as H+ acceptors such as acetaldehyde, acetone, pyruvic acid , and other carbonyl compounds , stimulates anaerobic fermentation. Small amounts of oxygen also stimulate fermentation <ref name="yakobson_introduction">[http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad. The Brettanomyces Project. Introduction. Retrieved 8/11/2015.]</ref>. 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 <ref name="Smith_2018" />.  ''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). <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref><ref name="Kumara_1993">[http://aem.asm.org/content/59/8/2352.short Localization and Characterization of α-Glucosidase Activity in Brettanomyces lambicus. H. M. C. Shantha Kumara, S. De Cort and H. Verachtert. 1993.]</ref> 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. <ref name="Steensels"></ref><ref>[http://www.scribd.com/doc/277758178/Insight-into-the-Dekkera-anomala-YV396-genome Insight into the Dekkera anomala YV396 genome. Samuel Aeschlimann. Self-published on Eureka Brewing Blog. Spet 2015.]</ref>.

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'' strains may possess both alpha an achieve in beers such as lambic and betaAmerican sour beers <ref name="yakobson_introduction"></ref><ref name="smith_divol_2016"></ref>. Alpha-glucosidases. These are the enzymes that allow ''Brettanomyces'' strains them to break down a broad range of sugarsmaltose, including long-chain carbohydrate molecules (polysaccharidesturanose, dextrinsmelezitose, and cellulose/cellobiose)trehalose, as well as dextrins such as maltotetraose and to liberate glycosidically bound 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 which occurs outside of the cell and may benefit other microorganisms if present such as lactic acid bacteria. These dextrins are unfermentable to left over after a normal ''Saccharomyces'' yeasts. fermentation <ref name="Steensels"></ref>. Some other polysaccharides can be fermented by ''Brettanomyces'', including starch, laminarin, and pectin <refname="Crauwels1">[http:<//wwwref>.scribd The more complex the starch or sugar, the slower it is hydrolyzed by the alpha-glucosidase enzymes.com/doc/277758178/Insight-into- The optimal pH for thealpha-Dekkeraglucosidase enzyme produced by one strain of ''B. bruxellensis'' was 6 and at a temperature of 39-anomala40°C (102-YV396-genome Insight into 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 Dekkera anomala YV396 genomeenzyme was not compared to other acids), which might contribute to slower ''Brettanomyces'' fermentation in acidic beers <ref name="Kumara_1993" />. Samuel Aeschlimann. SelfA 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-published on Eureka Brewing Blogglucosidase is not functional or poor in some strains. Spet 2015.] Additionally, when maltose is present instead of just glucose, the researchers saw an increased lag during the growth phase <ref name="colomer_2020_genome" /ref>.

Extracellular and intracellular alpha-glucosidase activity has been shown to break down sugars up to 9-12 chain carbons in one strain of ''B. lambicusbruxellensis'' (now classified as and ''B. bruxellensisnanus'')can also produce oligo-1,6-glucosidase, which is partly responsible for hydrolyze the slowalpha-1, over6 linkages in starch and glycogen to produce oligosaccharides, and then further break down these oligosaccharides to produce sugars with alpha-attenuation of wort that some strains 1,4 linkages (for example, maltose, in the case of ''Brettanomyces'' an achieve in beers such as lambic and American sour beers starches from malted barley <ref name="yakobson_introduction"><[https://www.ncbi.nlm.nih.gov/books/NBK22396/ref><ref name="smith_divol_2016">Complex Carbohydrates Are Formed by Linkage of Monosaccharides. Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. 2002.]</ref>). AlphaThese alpha-glucosidases 1,4 linkages (maltose) 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 by the case of extracellular alpha-glucosidase activity, this breakdown of complex sugars occurs outside maltose enzyme by strains of the cell and may benefit other microorganisms if present such as lactic acid bacteria. These dextrins are left over after a normal ''SaccharomycesBrettanomyces'' fermentation or other present microbes that produce this enzyme <ref name="SteenselsCrauwels_2014"></ref>. Some other polysaccharides can be fermented by Unlike for some domesticated diastatic ''S. cerevisiae'' strains, this ability for ''Brettanomyces'', including starch, laminarin, and pectin to break down starches has occurred in the wild without domestication <ref name="Crauwels1roach_2019"/></ref>"Amylases". The more complex the starch or sugarH. Taniguchi, the slower it is hydrolyzed by the alpha-glucosidase enzymesY. The optimal pH for the alpha-glucosidase enzyme produced by one strain Honnda, in Encyclopedia of ''B. bruxellensis'' was 6 and at a temperature of 39-40°C Microbiology (102-104°FThird Edition), and its activity was greatly reduced below a pH of 42009.5 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 ]</ref><ref name="Kumara_1993">[httphttps://aemwww.asmfacebook.orgcom/groups/contentMilkTheFunk/59permalink/83049768678384659/2352Ben Sykes.short Localization and Characterization of α-Glucosidase Activity in Milk The Funk Facebook post on ''Brettanomyces lambicus'' evolution to ferment dextrins. H. M. C. Shantha Kumara, S. De Cort and H. Verachtert. 199311/13/2019.]</ref>.

There is a highly genetic diversity between strains of ''Brettanomyces'' species, both in a [http://www.diffen.com/difference/Genotype_vs_Phenotype genotypic and phenotypic] sense <ref name="Crauwels1">[http://link.springer.com/article/10.1007/s00253-015-6769-9 Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains. S. Crauwels, A. Van Assche, R. de Jonge, A. R. Borneman, C. Verreth, P. Troels, G. De Samblanx, K. Marchal, Y. Van de Peer, K. A. Willems, K. J. Verstrepen, C. D. Curtin, B. Lievens. 2015]</ref>. 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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1279884332039778/ Lance Shaner experiment comparing the growth of various ''Brettanomyces spp'' on different growth mediums. 04/07/2016.]</ref><ref name="ncyc_searchbrett">[https://catalogue.ncyc.co.uk/catalogsearch/result/?q=brettanomyces National Collection of Yeast Cultures. Search for ''Brettanomyces''. Retrieved 04/07/2016.]</ref>. For example, some strains are not able to ferment maltose (often ''B. anomalus'' strains), which is almost half the sugar content of wort <ref>[https://eurekabrewing.wordpress.com/tag/sugar/ "Sugar composition of wort". Eureka Brewing Blog. Jan 13, 2015. Retrieved 04/07/2016.]</ref><ref>[https://www.pnas.org/doi/abs/10.1073/pnas.1105430108 Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast. Diego Libkind, Chris Todd Hittinger, Elisabete Valério, Carla Gonçalves, Jim Dover, Mark Johnston, Paula Gonçalves, and José Paulo Sampaio. DOI: https://doi.org/10.1073/pnas.1105430108. 2011.] See also [https://www.facebook.com/groups/MilkTheFunk/posts/7415625611798922/?comment_id=7427190037309146 this MTF thread]</ref>. Such strains would not be a good choice for [[100%25_Brettanomyces_Fermentation|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 from (wine barrels). Further studies are needed to see if this is a trend throughout the species <ref name="Crauwels1"></ref>. Daenen et al. (2007) found that none of the ''B. bruxellensis'' strains isolated from lambic that they tested could utilize cellobiose (see [[Brettanomyces#Glycosides_and_Beta-Glucosidase_Activity|glycosides]] below). This data point challenge 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 isoalted 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 <ref name="Tyrawa_2017">[https://onlinelibrary.wiley.com/doi/abs/10.1002/jib.565 The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations. Caroline Tyrawa Richard Preiss Meagan Armstrong George van der Merwe. 2019. DOI: https://doi.org/10.1002/jib.565.] See also: [https://www.facebook.com/groups/MilkTheFunk/permalink/1285391951489016/ "Funky can be Great: Brettanomyces bruxellensis Beer Fermentations" (poster for study). Caroline Tyrawa, Richard Preiss, and George van der Merwe. 2017.] </ref>. 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 <ref name="colomer_2020_genome" />.

Currently, research into how well ''Brettanomyces'' strains ferment the trisaccharide maltotriose has not been explored much by science, however. 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 <ref>[http://www.asbcnet.org/events/archives/2015Meeting/proceedings/Pages/54.aspx Determination of sugar metabolism profiles of non-traditional yeasts in the Saccharomyces and Brettanomyces families. J. D. Cook, W. A. DEUTSCHMAN. ASBC Proceeding. 2015.]</ref><ref name="Crauwels1"></ref>.

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, however one study by Tyrawa et al. found that several strains of ''B. bruxellensis'' fermented at 30°C "smelled terrible" of aromas typical of sulfur and autolysis <ref name=" Tyrawa_2017" /><ref>[https://www.facebook.com/groups/MilkTheFunk/posts/5684564058238428/?comment_id=5684866488208185&reply_comment_id=5692967664064734 Richard Preiss on very warm fermentation temperatures for ''Brettanomyces''. Milk The Funk Facebook group. 04/13/2022.]</ref>. 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) <ref name="Conterno_2006">[http://www.ajevonline.org/content/57/2/139 Genetic and Physiological Characterization of Brettanomyces bruxellensis Strains Isolated from Wines. Lorenza Conterno, C.M. Lucy Joseph, Torey J. Arvik, Thomas Henick-Kling, Linda F. Bisson. 2006.]</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/24290676 Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in Wine. Zuehlke JM, Edwards CG. 2013. DOI: 10.4315/0362-028X.JFP-13-243R.]</ref> 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) <ref name="Tyrawa_2017" />. At a temperature of 35°C (95°F), fermentation is greatly inhibited due to cell death for most strainsof ''B. bruxellensis'', with about a third of strains able to grow as high as 37°C (98.6°F) <ref name="Conterno_2006" />, and complete elimination in wines at 50°C for 5 minutes (see also [[Barrel#Sanitizing|Barrel Sanitizing]] and [[Quality_Assurance#Pasteurization|Pasteurization]]) <ref name="Couto_2005">[https://pubmed.ncbi.nlm.nih.gov/15996781/ Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces. José António Couto, Filipe Neves, Francisco Campos, Tim Hogg. 2005. DOI: 10.1016/j.ijfoodmicro.2005.03.014.]</ref><ref name="Nunes de Lima 2020">[https://www.sciencedirect.com/science/article/abs/pii/S0740002020302069 Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Adriana Nunes de Lima, Rui Magalhães, Francisco Manuel Campos, José António Couto. 2020. DOI: https://doi.org/10.1016/j.fm.2020.103617.]</ref>. ''B. naardenensis'' is less tolerant to extreme temperatures, and it has been demonstrated that this species cannot grow at 30°C or higher <ref name="Tiukova_2019" />. 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) <ref name="Brandam_2008" />. 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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1555689637792578/ MTF conversation with Richard Preiss of Escarpment Labs. 01/20/2017.]</ref>. For more information on how fermentation temperature affects the flavor compounds of 100% ''Brettanomyces'' fermentation, see [[100%25_Brettanomyces_Fermentation#Impact_of_Fermentation_Temperature|Impact of Fermentation Temperature]].

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) <ref>[https://en.wikipedia.org/wiki/Glycoside "Glycoside." Wikipedia. Retrieved 06/27/2016.]</ref>. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic <ref name="Daenen2">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00421.x/pdf Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. Luk Daenen, Femke Sterckx, Freddy R. Delvaux, Hubert Verachtert & Guy Derdelinckx. 2007.]</ref>. It is speculated that flavor compounds from hops can also be released from glycosides <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref>, ; 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 <ref name="Vervoort">[http://onlinelibrary.wiley.com/wol1/doi/10.1111/jam.13200/abstract Characterization of the recombinant Brettanomyces anomalus β-glucosidase and its potential for bioflavoring. Yannick Vervoort, Beatriz Herrera-Malaver, Stijn Mertens, Victor Guadalupe Medina, Jorge Duitama, Lotte Michiels, Guy Derdelinckx, Karin Voordeckers, and Kevin J. Verstrepen. 2016.]</ref>.  Beta-glucosidase also allows the breakdown of cellobiose and cellotriose <ref>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC241500/ Fermentation of Cellodextrins by Different Yeast Strains. Pierre Gondé, Bruno Blondin, Marc Leclerc, Robert Ratomahenina, Alain Arnaud, and Pierre Galzy. 1984.]</ref><ref name="Roos_2018">[https://www.ncbi.nlm.nih.gov/pubmed/30246252?dopt=Abstract Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>. 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 <ref name="Daenen1" /><ref name="Roos_2018" />. Additionally, a study by Tyrawa et al. from [[Escarpment Laboratories]] agreed that wine isolated strains were generally better at fermenting cellobiose than strains isoalted isolated from beer at 15°C (59°F), however. 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 <ref name="Tyrawa_2017" />, and the [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|table from the NCYC ''Brettanomyces'' strains]] suggests that fermenting cellobiose is generally rare for ''B. bruxellensis''. This also suggests that not only is are ''B. bruxellensis'' strains that are isolated from beer are 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).

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 <ref name="Spaepen"></ref>. 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) <ref name="Tyrawa_2017" />.

[https://www.britannica.com/science/phenol 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 found present in the non-starch polysaccharide arabinoxylan in malt 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) <ref name="kalb_2021">[https://pubs.acs.org/doi/full/10.1021/acs.jafc.1c03018 Investigations into the Ability to Reduce Cinnamic Acid as Undesired Precursor of Toxicologically Relevant Styrene in Wort by Different Barley to Wheat Ratios (Grain Bill) during Mashing. Valerian Kalb, Torsten Seewald, Thomas Hofmann, and Michael Granvogl. Journal of Agricultural and Food Chemistry 2021 69 (32), 9443-9450. DOI: 10.1021/acs.jafc.1c03018.]</ref><ref name="lentz_2018">[http://www.mdpi.com/2311-5637/4/1/20/html#B13-fermentation-04-00020 The Impact of Simple Phenolic Compounds on Beer Aroma and Flavor. Michael Lentz. 2018. doi: 10.3390/fermentation4010020.]</ref>. 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) <ref>[http://www.ajevonline.org/content/early/2018/05/02/ajev.2018.17092 Influence of Oenococcus oeni and Brettanomyces bruxellensis on Wine Microbial Taxonomic and Functional Potential Profile. Marie Lisandra Zepeda-Mendoza, Nathalia Kruse Edwards, Mikkel Gulmann Madsen, Martin Abel-Kistrup, Lara Puetz, Thomas Sicheritz-Ponten, Jan H. Swiegers, Am J Enol Vitic. May 2018. DOI: 10.5344/ajev.2018.17092.]</ref>. 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 <ref>[https://pubs.acs.org/doi/full/10.1021/jf204908s Hydroxycinnamic Acid Ethyl Esters as Precursors to Ethylphenols in Wine. Josh L. Hixson, Nicola R. Sleep, Dimitra L. Capone, Gordon M. Elsey, Christopher D. Curtin, Mark A. Sefton, and Dennis K. Taylor. 2012. DOI: 10.1021/jf204908s.]</ref>. 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 <ref name="Cortese_2020">[https://www.sciencedirect.com/science/article/pii/S0021967319310295 Quantification of phenolic compounds in different types of crafts beers, worts, starting and spent ingredients by liquid chromatography-tandem mass spectrometry. Manuela Cortese, Maria Rosa Gigliobianco, Dolores Vargas Peregrina, Gianni Sagratini, Roberta Censi, Piera Di Martino. Journal of Chromatography A; Volume 1612, 8 February 2020, 460622. DOI: https://doi.org/10.1016/j.chroma.2019.460622.]</ref>. 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 <ref>[https://link.springer.com/article/10.1007/s10482-016-0793-3 González, C., Godoy, L. & Ganga, M.A. Identification of a second PAD1 in Brettanomyces bruxellensis LAMAP2480. Antonie van Leeuwenhoek 110, 291–296 (2017). https://doi.org/10.1007/s10482-016-0793-3.]</ref><ref name="Lentz">[http://www.mdpi.com/2304-8158/4/4/581/htm Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast. Michael Lentz and Chad Harris. 2015.]</ref><ref>[https://www.biorxiv.org/content/10.1101/2024.04.16.586637v1 Characterization of Brettanomyces bruxellensis phenolic acid decarboxylase enzyme expressed in E. coli. Michael R. Lentz. bioRxiv 2024.04.16.586637; doi: https://doi.org/10.1101/2024.04.16.586637.]</ref>, ''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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1632316743463200/ Analysis of phenolic acid decarboxylase enzyme from the wine spoilage yeast Brettanomyces bruxellensis (poster). Mike Lentz, Jamie Lynch, Pricilla Walters, Rachel Licea, Henok Daniel, Kimberly Pereira. 2017.]</ref>. 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'' <ref name="Tyrawa_Masters">[https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation. University of Guelph, Masters Thesis. Department of Molecular and Cellular Biology. 2020.]</ref>. 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 <ref name="colomer_2020_genome" />.

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 <ref name="Barata_2008">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00415.x/full The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine. André Barata, Daniela Pagliara, Tiziana Piccininno, Francesco Tarantino, Wilma Ciardulli, Manuel Malfeito-Ferreira, Virgílio Loureiro. 2008. DOI: 10.1111/j.1567-1364.2008.00415.x]</ref>, some data supports that pitching rate does not have an effect on how much phenol content is produced by ''Brettanomyces''<ref name="MTF_Brett_Secondary">[http://www.milkthefunk.com/wiki/Brettanomyces_secondary_fermentation_experiment Brettanomyces secondary fermentation experiment. Milk The Funk Wiki. Lance Shaner and Richard Preiss. Retrieved 04/21/2016.]</ref>. 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 <ref>[https://www.ncbi.nlm.nih.gov/pubmed/24010603 Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma. Curtin CD, Langhans G, Henschke PA, Grbin PR. 2013]</ref>. [https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert's Bachelors thesis] also reported no correlation between fermentation rate and phenol production in several strains of ''B. bruxellensis'' <ref name=Humbert_2021">[https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert for the degree of Honors Baccalaureate of Science in Chemical Engineering presented on May 21, 2021. Title: Performance of Brettanomyces Yeast Strains in Primary and Secondary Beer Fermentations.]</ref>. 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 [[Brettanomyces#Ester_Production|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 <ref name="Tyrawa_2017" />. 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 <ref name=Humbert_2021" />.

It has been hypothesized that the production and destruction of various phenols by ''Brettanomyces'' is connected with the [https://en.wikipedia.org/wiki/Redox 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 <ref>[http://www.sciencedirect.com/science/article/pii/S0960982214016558 Olfaction: Smells Like Fly Food. Geraldine A. Wright. 2015.]</ref><ref name="smith_divol_2016"></ref>. Phenols have been shown to have positive effects on decreasing protein glycation, a complication associated with type 2 diabetes <ref>[http://www.asbcnet.org/publications/journal/vol/2017/Pages/ASBCJ-2017-1323-01.aspx High Phenolic Beer Inhibits Protein Glycation In Vitro. Susan M. Elrod, Phillip Greenspan, Erik H. Hofmeister. 2017. ]</ref>.

fermentations with respect to time. Presentation at 2008 NHC. pg 12.</ref> <ref name="Yakobson_Michigan">[http://www.mbaa.com/districts/michigan/events/Documents/2011_01_14BrettanomycesBrewing.pdf Yakobson, Chad]. Brettanomyces in Brewing the horse the goat and the barnyard. 1/14/2011</ref> (Musty, Medicinal, Band-aid, Plastic) || Vinyl phenol || p-Coumaric Acid || 0.2 ppm (flavor; in beer) <ref>[http://www.scielo.br/scielo.php?pid=S1516-89132013000600018&script=sci_arttext Determination of 4-vinylgaiacol and 4-vinylphenol in top-fermented wheat beers by isocratic high performance liquid chromatography with ultraviolet detector. Mingguang Zhu; Yunqian Cui. Dec 2013.]</ref> || C₈H₈O <ref name="goodscents_4VP">[http://www.thegoodscentscompany.com/data/rw1005801.html The Good Scents Company. 4-Vinylphenol. Retrieved 08/18/2015.]</ref> || Production level is different across species/strains of ''Brettanomyces'' <ref name="Oelofse">[http://www.sciencedirect.com/science/article/pii/S0740002008002050 Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production. A. Oelofse, A. Lonvaud-Funel, M. du Toit. 2009.]</ref>. 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 <ref name="Cortese_2020" />. Coumaric levels are generally higher in barley malt than they are in wheat malt. Coumaric acid is stable through the wort boiling process <ref name="kalb_2021" />.  It's also been demonstrated that the presence of p-coumaric can assist in reviving so-called [[Quality_Assurance#Viable_But_Nonculturable|VNBC cells of ''B. bruxellensis'']], suggesting that ''Brettanomyces'' can use energy sources such as p-coumaric acid to maintain survival in nutrient poor conditions <ref>[https://www.mdpi.com/2306-5710/9/3/69 Chandra M, Branco P, Prista C, Malfeito-Ferreira M. Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis. Beverages. 2023; 9(3):69. https://doi.org/10.3390/beverages9030069.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/posts/7289633364398148/?comment_id=7295436620484489 Richard Preiss. Milk The Funk Facebook group thread about p-coumaric acid metabolism. 08/27/2023.]</ref><ref>[https://www.mdpi.com/2306-5710/9/3/69 Chandra M, Branco P, Prista C, Malfeito-Ferreira M. Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis. Beverages. 2023; 9(3):69. https://doi.org/10.3390/beverages9030069.]</ref>.

| 4-Vinylguaiacol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Clove) || Vinyl phenol || Ferulic Acid || 0.3 ppm (flavor; in beer) <ref>[http://www.aroxa.com/beer/beer-flavour-standard/4-vinyl-guaiacol/ Aroxa Website. 4-Vinylguaiacol. Retrieved 08/19/2015.]</ref> || C₉H₁₀O₂. Also known as 2-methoxy-4-vinyl phenol <ref name="goodscents_4VG">[http://www.thegoodscentscompany.com/data/rw1005101.html The Good Scents Company. 2-methoxy-4-vinyl phenol. Retrieved 08/18/2015.]</ref> . || C₉H₁₀O₂ <ref name="goodscents_4VG"></ref>. Produced by some strains of ''S. cerevisiae'' (see [[Saccharomyces#Phenolic_Off_Flavor_Strains|''Saccharomyces'']]) <ref name="Coghe_2014">[http://pubs.acs.org/doi/abs/10.1021/jf0346556 Ferulic Acid Release and 4-Vinylguaiacol Formation during Brewing and Fermentation:  Indications for Feruloyl Esterase Activity in Saccharomyces cerevisiae. Stefan Coghe, Koen Benoot, Filip Delvaux, Bart Vanderhaegen, and Freddy R. Delvaux. 2004.]</ref>. Some ''Brettanomyces'' species/strains may also be able to produce this compound at varying levels <ref name="Joseph"></ref><ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1995.tb07374.x/abstract The biotransformation of simple phenolic compounds by Brettanomyces anomalus. Duncan A.N. Edlin1, Arjan Narbad, J. Richard Dickinson1 andDavid Lloyd. 2006.]</ref><ref name="Oelofse"></ref>. Organic malts have been linked to higher levels of 4VG, vanillan, and their malt precursor ferulic acid <ref name="Iyuke_2008">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.2008.tb00773.x/full The Effect of Hydroxycinnamic Acids and Volatile Phenols on Beer Quality. S. E. Iyuke, E. M. Madigoe, and R. Maponya. 2008.]</ref>. 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) <ref name="Coghe_2014" /><ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/j.2050-0416.2000.tb00036.x Extraction and Assay of Ferulic Acid Esterase From Malted Barley. F. J. Humberstone D. E. Briggs. 2012.]</ref>. 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 sudies studies showing that barley malt often contains more ferulic acid than wheat malt (see [https://www.facebook.com/groups/MilkTheFunk/permalink/2053354874692716/ this MTF thread] that explains why this is) <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1002/jib.189 Enhancing the levels of 4‐vinylguaiacol and 4‐vinylphenol in pilot‐scale top‐fermented wheat beers by response surface methodology. Yunqian Cui, Aiping Wang, Zhuo Zhang, R. Alex. Speers. 2005. DOI: https://doi.org/10.1002/jib.189.]</ref><ref name="Coghe_2014" /><ref name="lentz_2018" /><ref>[https://www.sciencedirect.com/science/article/pii/S0308814608003348 Release of phenolic flavour precursors during wort production: Influence of process parameters and grist composition on ferulic acid release during brewing. Nele Vanbeneden, Tom Van Roey, Filip Willems, Filip Delvaux, Freddy R.Delvaux. 2008. https://doi.org/10.1016/j.foodchem.2008.03.029]</ref><ref>[https://pdfs.semanticscholar.org/74cd/c0ad3811d95b92c1ecb55ddea392de95ba59.pdf Ferulic Acid in Cereals – a Review. Hüseyin BOZ. 2015. doi: 10.17221/401/2014-CJFS.]</ref>. A more recent studies disagreed and found a linear increase soluble ferulic acid correlated with higher percentages of wheat malt <ref name="kalb_2021" />. 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 <ref name="kalb_2021" />. 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) <ref>[https://www.mdpi.com/2076-3921/10/7/1124 Shopska V, Denkova-Kostova R, Dzhivoderova-Zarcheva M, Teneva D, Denev P, Kostov G. Comparative Study on Phenolic Content and Antioxidant Activity of Different Malt Types. Antioxidants. 2021; 10(7):1124. https://doi.org/10.3390/antiox10071124.]</ref>. Ferulic acid is stable through the wort boiling process <ref name="kalb_2021" />.

| 4-Vinylcatechol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Plastic, Bitter, Smokey) || Vinyl phenol || Caffeic Acid || || C₈H₈O₂ <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/441226 PubChem. 3-Vinylcatechol. Retrieved 08/18/2015.]</ref> || Production level is difference across species/strains of ''Brettanomyces'' <ref name="Oelofse"></ref>.

In the presence of oxygen, ''Brettanomyces'' species produce acetic acid as a byproduct of glucose fermentationrespiratory metabolism. The more oxygen that is present, the more acetic acid is produced and the less ethanol is produced by ''Brettanomyces'' <ref>[https://link.springer.com/article/10.1007/s00253-002-1197-z Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. M. G. Aguilar Uscanga, M.L. Délia, P. Strehaiano. 2003.]</ref><ref>[https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain," Escarpment Labs blog post, 01/20/21.]</ref><ref name="Rozpędowska" /><ref>[https://academic.oup.com/femsyr/article/13/1/34/544881?login=true Fernanda Cristina Bezerra Leite, Thiago Olitta Basso, Will de Barros Pita, Andreas Karoly Gombert, Diogo Ardaillon Simões, Marcos Antonio de Morais, Jr, Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants, FEMS Yeast Research, Volume 13, Issue 1, February 2013, Pages 34–43, https://doi.org/10.1111/j.1567-1364.2012.12007.x]</ref><ref>[https://link.springer.com/article/10.1007/BF00400180 Wijsman, M.R., van Dijken, J.P., van Kleeff, B.H.A. et al. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie van Leeuwenhoek 50, 183–192 (1984). https://doi.org/10.1007/BF00400180.]</ref>. In an environment with oxygen present, ''Brettanomyes'' switches to respiratory metabolism. 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). The acetate that is produced by ''Brettanomyces'' under aerobic conditions is an important requirement for the cells to fully metabolize certain types of sugars like galactose <ref>[https://link.springer.com/article/10.1007/s12010-023-04398-w Teles, G.H., Xavier, M.R., Da Silva, J.M. et al. The Metabolism of Respiring Carbon Sources by Dekkera bruxellensis and Its Relation with the Production of Acetate. Appl Biochem Biotechnol (2023). https://doi.org/10.1007/s12010-023-04398-w.]</ref>. 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) <ref>[https://link.springer.com/article/10.1023/A:1022592810405 Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH. S.N. FreerB. DienS. Matsuda. 2003.]</ref><ref name="Hubbe"></ref>. 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 [[Mixed_Fermentation#Aging|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'' <ref name="colomer_2020_genome" /><ref name="Tiukova_2019" />. Acetic acid produced by ''Brettanomyces'' is also used in the synthesis of [[Brettanomyces#Ester_Production|acetate esters]] such as ethyl acetate, perhaps as a mechanism to protect itself after hindering other microbes via the acetic acid precursor. ''Brettanomyces'' is not known to produce significant amounts of lactic acid.  ''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) <ref name="yakobson1"></ref>. 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 <ref name="Smith_2018" />.

===[[Bootleg Biology]]/[[Spot Yeast]]===

| 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 <ref name="coffey_drei">[http://www.alesoftheriverwards.com/2015/12/brettanomyces-drei-vs-brettanomyces-vrai.html Ed Coffey. "Brettanomyces Drei vs. Brettanomyces Vrai". Ale of the Riverwards blog. 12/07/2015. Retrieved 11/13/2018.]</ref>. '''Commercial pitches only.'''

| 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 <ref name="escarpment_strains">[http://www.escarpmentlabs.com/strains "Strains" list. Escarpment Laboratories website. Retrieved 12/07/2017.]</ref>. ||Isolated from a bottle of Orval <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1278416635519881/?comment_id=1279065935454951&reply_comment_id=3665429823485205 Richard Preiss. Milk The Funk Facebook group post about Escarpment Labs Brett B culture.. 07/11/2020.]</ref>.

===[[Fermmento Labs]] (Brazil- CLOSED)===

===[[GigaYeast]](CLOSED)===

===[[Jasper Yeast LLC]]===

| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY033 - Brett brux Amsterdam || Slow growing, good for conditioning in the style of Orval || Amsterdam, Netherlands <ref name="jasper_brett"Chateaux>[https://jasperyeast.com/yeast/brettanomyces "Brettanomyces". Jasper Yeast Website.]</ref>|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY044 - Brett brux Rosa || Comparable to JY033, but slightly more funky, tart and aggressive. || California, USA <ref name="jasper_brett" />|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY87 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.<ref name="jasper_brett"/>|-| 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 <ref name="jasper_brett"/>|-| 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 <ref name="jasper_brett"/>|-| 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 <ref name="jasper_brett"/>|-

===[[Mainiacal Yeast]](CLOSED)===

| 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 ToelranceTolerance: medium-high, compares to WLP645. Can ferment lactose <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2455742991120567/?comment_id=2468302423197957&reply_comment_id=2471170252911174&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Adi Hastings from Omega Yeast Labs. Milk The Funk Facebook thread on ''Brettanomyces'' strains that can ferment lactose. 01/19/2019.]</ref>. 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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3293190840709107/?comment_id=3293807780647413 Lance Shaner. Milk The Funk Facebook group thread on OYL-201 attenuation. 02/21/2020.]</ref>. ||

''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|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#Brettanomyces_Metabolism|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.

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 [[Brettanomyces_Fermentation|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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1099473923414154/?comment_id=1099522943409252&offset=0&total_comments=25&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Richard Preiss of Escarpment Yeast Labs on MTF. 6/26/2015.]</ref>. 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.

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 <ref name="Yakobson_Propagation">[http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-results/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Results. Retrieved 2/17/2015]</ref><ref name="MarkTrent">[https://www.facebook.com/groups/MilkTheFunk/permalink/1114254011936145/ Conversation with Mark Trent and Lance Shaner on MTF regarding Brett pitching rates. 07-21-2015.]</ref>. 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 [[Brettanomyces_Fermentation|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) <ref name="MarkTrent"></ref>.

====MYPG Growth Substrateand Other Laboratory Substrates====

Major yeast labs will often store yeast in a -80°C laboratory freezer in a media/glycerol solution, however, although this option is generally not practical for brewers <ref>[https://en.wikipedia.org/wiki/Cryopreservation Cryopreservation. Wikipedia. Retrieved 07/07/2016.]</ref>. The next best option for long-term storage of ''Brettanomyces'' is freezing with 10% glycerol in a home freezer, however. 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, [http://suigenerisbrewing.blogspot.com/2015/11/easy-home-yeast-banking-and-video.html 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 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1083075848387295/?comment_id=1083272091701004&offset=0&total_comments=13&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Matt Humbard, Ritchie Preiss, and Jeff Melo on MTF. 6/4/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1115817201779826&offset=0&total_comments=34&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Nick Impellitteri on MTF regarding storing Brett on agar plates. 7/24/2015.]</ref>.

Occasional feeding has been shown to keep ''Brettanomyces'' alive in beer for brewers who do not have a lab, ; however, many variables may come into play as far as how effective this will be for individual strains and in different environments. Although no research has been done to indicate what the best practices are for feeding ''Brettanomyces'' to keep it alive in beer, we recommend trying this method: every 3-6 months swirl the vessel so as to suspend all of the yeast and then decant 70-90% of the beer and suspended yeast slurry, and replace it with a 1.040 starter wort with yeast nutrients. This method will discard a lot of the old yeast cells, while retaining enough living cells for replication <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1345924532102424/?comment_id=1345979272096950&reply_comment_id=1346020438759500&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Mark Trent and Richard Preiss on MTF regarding occasional feeding of ''Brettanomyces'' to keep it alive. 07/07/2016.]</ref>. Some strains may survive extended periods of aging in beer, ; however, their viability and vitality will be greatly reduced over time. Interestingly, ''Brettanomyces'' remains '''more''' viable over time if it was co-fermented with ''S. cerevisiae'' than if it was fermented without the presence of ''S. cerevisiae''; i.e. [[100%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] or ''Brettanomyces'' and ''Lactobacillus <ref name="Hubbe" />.

* [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]