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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, pale ales, stone fruitsporters, 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 Span 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'' is commonly isolated from has been thought to occur naturally on the surface skins of wood structures within breweriesfruit such as apples and grapes. However, wineriesthere are only a handful of reports of ''Brettanomyces'' being identified on the skins of fruit, and sometimes cideriesin 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. These include structures such as wooden fermentation vesselsand Luman, walls of the buildingE. (2014), as well as the inside surface Genetic and physiological characterization of wood barrels yeast isolated from ripe fruit and actually buried within the wood analysis of barrels up to 8 mm fermentation and brewing potential, J. Inst. Brew., 120: 559– 564. DOI: 10.1002/jib.154.]</ref><ref name="Agnolucci_2017Comitini" >[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, with a spoilage wine yeast, on the highest concentration surface of surviving cells being at the top staves where oxygen is more accessiblegrape berries. Vincent Renouf, Aline Lonvaud-Funel. Some strains are able to utilize the cellulose of the wood as a carbon source2007. Ozone has been shown to be an effective way to kill 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 is buried in the wood of oak barrels, but processing equipment may be the ozone must be applied for an adequate time to allow primary source for the ozone to diffuse into the oak''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 In addition, ''Brettanomyces'' within has been isolated in abundance from the wood surfaces of barrels equipment/processed materials in wineries and breweries <refname="smith_divol_2016" /><ref name="Schifferdecker" /><ref name="Loureiro_2003">[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation 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 wine spoilage yeast Dekkera bruxellensis by hot water treatmentin wines and inhibitory effect of sulphur dioxide. Fabriziof Barata A, VigentiniCaldeira J, ParisiBotelheiro R,PicozziPagliara D, CompagnoMalfeito-Ferreira M, FoschinoLoureiro V. 20152008.]</ref>(Table 1). Although the role 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'' appears to be limited in distillation, it has been isolated during the fermentation process wild (so far they have only surveyed non-human inhabited wild areas of tequila makingthe 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 has is therefore unclear that ''Brettanomyces'' found on grape skins originated there or from the industrial processing where it is more abundant. It is also been isolated from drainsthought to disperse via fruit-flies (called "vectors" in the scientific literature), pumps, transfer hosessimilar to how ''Saccharomyces'' travels, although direct evidence for this has been reported rarely and other only on fruit-flies in wineries that are likely to come into contact with equipment /processed material that is difficult to sanitize. The survivability of already contaminated with ''Brettanomyces'' has also partly been attributed to its ability to form a <ref>[https://enyoutu.be/G2nhUM5PIrg?t=309 Dr. Bryan Heit. BotB - Where (Do) The Wild Brettanomyces Roam?.wikipedia~5 mins in.orgRetrieved 07/wiki10/Biofilm biofilm2022.] (in particular </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" />. ''B. bruxellensisBrettanomyces''). Microorganisms that can form is known to be difficult to grow in a biofilm are more resistant lab due to chemical cleaning agents and sanitizers than those that donslow growth, specific nutrient requirements, or perhaps because of a "VBNC" state (see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild ''Brettanomyces''t. ]] for more information), which may account for the lack of evidence for fruit being the primary natural habitat for ''Brettanomyces'' has therefore been identified as a significant contaminate for breweries and wineries. Oak barrels from wineries with unsanitary practices, in particularMore recently, techniques have been identified as common contamination sites for invented to more easily isolate and grow ''Brettanomyces''B<ref name="Renouf_2007" /><ref name="Comitini" />. bruxellensisThere 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'' is also commonly have been found in sherrythe root systems of dill, common beans, sunflowers, maize, corn, jute, cassava, and is 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. (although only rarely2016) , Arbuscular mycorrhizal fungi induced changes in olive productionrhizosphere, lemonadeessential oil and mineral nutrients uptake in dill/common bean intercropping system. Ann Appl Biol, kombucha169: 384-397. https://doi.org/10.1111/aab.12309.]</ref><ref>[https://archive.aessweb.com/index.php/5003/article/view/3333 I.O, yogurtS. ., & G.P, O. . (2012). Diversity of Fungal Populations in Soils Cultivated With Cassava Cultivar TMS 98/0505. Journal of Asian Scientific Research, pickles2(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 soft drinksnon-rhizosphere soil mycoflora of Corchorus olitorius (Jute). G. ''BS. 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. anomalus'' International Journal of Biosciences and ''BBiotechnology, p. 12-24, dec. 2017. ISSN 2655-9994. doi: https://doi.org/10.24843/IJBB.2017.v05.i01.p02.]</ref><ref>[https://www.sciencedirect. bruxellensis'' are generally found much more commonly than the other three species 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 ''Brettanomyces''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 name="smith_divol_2016">[httphttps://www.sciencedirect.com/science/article/abs/pii/S0740002016302659 Brettanomyces bruxellensisS1049964419303238 Nivien A. Nafady, Mohamed Hashem, Elhagag A. Hassan, Hoda A.M. Ahmed, a survivalist prepared for the wine apocalypse Saad A. Alamri. The combined effect of arbuscular mycorrhizae and other beveragesplant-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. Brendan Dphp/sains_seni/article/view/5613 Isolation and Characterization of Yeast from Rhizosphere Avicennia Marina Wonorejo. SmithSitatun Zunaidah, Benoit DivolNur Hidayatul Alami. 2014. DOI: 10.12962/j23373520. June 2016v3i1.5613.]</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]].

Unlike most genres The occurrence of yeast, ''Brettanomyces'' has the characteristics of being very tolerant to harsh conditionsbeen more commonly identified in industrial food processing areas (wine, beer, kombucha, soft drinks, dairy products, tea, sourdough, including high amounts of alcohol (up to 14etc.5-15% ABV ) <ref name="Crauwels1Crauwels_2016" /><ref name="Agnolucci_2017" />), a pH as low as 2 <ref>[httphttps://wwwacademic.winesandvinesoup.com/template.cfmfemsyr/article-abstract/17/1/fow105/2670560/Fermentation-assays-reveal-differences-in-sugar?sectionredirectedFrom=news&content=141954 Wines fulltext Fermentation assays reveal differences in sugar and Vines(off-) flavor metabolism across different Brettanomyces bruxellensis strains. New Research on Role of Yeast Fermentation assays reveal differences in Winemaking; report on a presentation by David Mills sugar and Lucy Joseph from UC Davis(off-) flavor metabolism across different Brettanomyces bruxellensis strains. 11/14/2014Sam 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. Retrieved 08/16/2015.2016]</ref>. For example, ''B bruxelensis'', ''B. anomala'', and environments with low nitrogen <ref name="Schifferdecker"></ref>''B. custersianus'' have mostly been isolated from wine or beer production, while ''B. It naardenensis'' has mostly 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 isolated from soda production <ref name="Smith_2017Tiukova_2019">[https://www.sciencedirectmdpi.com/science2076-2607/article7/pii11/S0740002017308249 The carbon consumption pattern 489 Assembly and Analysis of the Genome Sequence of the spoilage yeast Yeast Brettanomyces bruxellensis in synthetic wine-like mediumnaardenensis CBS 7540. Brendan DIevgeniia A. SmithTiukova, Huifeng Jiang, Jacques Dainat, Benoit DivolMarc P. Hoeppner, Henrik Lantz, Jure Piskur, Mats Sandgren, Jens Nielsen, Zhenglong Gu, and Volkmar Passoth. 2019. 2017DOI: https://doi.org/10.3390/microorganisms7110489.]</ref>. This capability allows ''Brettanomyces'' is not considered to survive be airborne; however, one study has demonstrated a very small amount of cells in alcoholic beverages such as beer, the air at wineries where wine, and cider. In alcoholic beverages, with ''B. bruxellensisBrettanomyces'' tends to lag after in it was being handled (most of the yeasts found in the primary fermentation with air were ''SaccharomycesAureobasidium''. It is believed that during this lag phase, and ''B. bruxellensisCryptococcus'' adapts to the harsh conditions of the beverage (low pH, high concentrations of ethanol, which aren't considered spoilage organisms in beer and limited sugar/nitrogen sourceswine). After this lag phase, This set of studies also determined that very specific methodology was needed in order capture ''B. bruxellensisBrettanomyces'' can grow from the air, and survive when no other yeasts canindicated that the yeast was "stressed". While it is possible for ''Brettanomyces'' is also more resistant to pH and temperature changesbe briefly carried by gusts of air, and tolerant of environments limited it only happens in oxygen (although the vicinity where the ''Brettanomyces'' prefers the availability 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 at least a little bit yeast mycoflora in winery air samples and their risk of oxygen)wine contamination. E. Ocón, P. Garijo, S. Sanz, C. Olarte, R. López, P. Santamaría, A.R. Gutiérrez. ScientificallyFood Control Volume 34, Issue 2, which specific nitrogen December 2013, Pages 261–267.]</ref>. Good cleaning and sanitation and carbon sources cold temperatures should be employed to keep ''B. bruxellensisBrettanomyces'' from contaminating other equipment; however, flying insects are also a potential cause for contamination of ''Brettanomyces'' uses in these stressful environments has not received much research <ref name="smith_divol_2016"></ref>(although evidence for this is lacking).

''Brettanomyces'' is known for not producing much glycerol 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 beerpreventing microbiological spoilage. Eur Food Res Technol 233, 285–291 (2011). https://doi.org/10.1007/s00217-011-1523-8.]</ref><ref>[https://enagris.wikipediafao.org/wikiagris-search/Glycerol Glycerol] is a colorlesssearch.do?recordID=IT2007601151 Fontanot, S.; Ninino, sweet-tastingM.E.; Comi, viscous liquid that is an important contributor to G.; Elimination of Dekkera/Brettanomyces from barriques of the mouthfeel Italian CDO Isonzo area. Controlled Designation of beerOrigin; Friuli-Venezia Giulia. 2006.]</ref>). Glycerol is produced These include structures such as a stress response by a wide range wooden fermentation vessels, walls of microbesthe building, including as well as the inside surface of wood barrels and actually buried within the wood of barrels. ''S. cerevisiaeBrettanomyces''has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and various species 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 strains of one study found that the ''DebaryomycesBrettanomyces''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 ''CandidaBrettanomyces''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 ''LachanceaBrettanomyces'', and on wine grapes. Liquid ozone has been shown to be less effective at eliminating ''ZygosaccharomycesBrettanomyces''. Despite not producing amounts Heating the inside of glycerol that are perceivable in beer, some strains 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 bruxellensis'' actually produce glycerol which is stored inside within the wood of their cells as a response to osmotic stress. They can also uptake glycerol into their cells. Doing so allows the cells to survive osmotic pressure barrels (see [[Barrel#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.sciencedirectncbi.nlm.nih.comgov/sciencepubmed/article/pii/S0740002013001251?via%3Dihub Osmotic stress response in the 25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensisby hot water treatment. Silvia GalafassiFabrizio, Marco ToscanoVigentini, Ileana VigentinParisi, Jure PiškurPicozzi, Concetta Compagno, Foschino. 20132015.]</ref><ref>[https://academicwww.oupsciencedirect.com/femslescience/advance-article-abstract/doipii/10.1093/femsle/fny020/4828327?redirectedFrom=fulltext Osmotolerance S1466856417310068 Control of Dekkera Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and the role of two Stl glycerol-proton symportersgaseous ozone. Jana ZemančíkováFrancesco Craveroa, Michala DuškováVasileios Englezos, Hana ElicharováKalliopi Rantsiou, Klára PapouškováFabrizio Torchio, Hana SychrováSimone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. It is currently not known how many strains are capable Although the role of producing glycerol internally''Brettanomyces'' appears to be limited in distillation, or if this amount it has been isolated during the fermentation process of glycerol tequila making. It has any impact on perceived mouthfeel 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 beer if [[Quality_Assurance#Biofilms|biofilm]] (in particular ''B. bruxellensis''). Microorganisms that can form a substantial amount of biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. ''Brettanomyces'' cells eventually autolyze (see [https://wwwhas therefore been identified as a significant contaminate for breweries and wineries.facebook Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''B.com/groups/MilkTheFunk/permalink/2003626776332193/ this MTF thread])bruxellensis''. The role of glycerol ''Brettanomyces'' is also commonly found in creating mouthfeel sherry, and is debatable 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 wine world other three species of ''Brettanomyces'' <refname="smith_divol_2016">[httpshttp://www.winesandvinessciencedirect.com/featuresscience/article/68760 Tim Pattersonpii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. "Many Roads to Mouthfeel"Brendan D. Wines & Vines MagazineSmith, Benoit Divol. Nov 2009. Retrieved 03/23/2018June 2016.]</ref>.

The genetic diversity Unlike most genera of yeast, ''Brettanomyces'' is particularly widehas 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. Some studies have indicated DOI: https://doi.org/10.1016/j.fm.2017.12.011.]</ref>. It has been reported that strains of ''B. bruxellensis'' have adapted is more tolerant of high levels of bicarbonate than compared to specific environments''S. cerevisiae'' (levels above 100 mg/l slow the fermentation of ''B. For examplebruxellensis'', but do not completely inhibit it, with up to 400 mg/l being tested in one study found ) <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'' isolated from wine had 20 genes involved in adapts to the harsh conditions of the metabolism beverage (low pH, high concentrations of carbon ethanol, and limited sugar/nitrogen, whereas strains isolated from beer did notsources). This indicated that After this lag phase, ''B. bruxellensis'' strains living 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 wine have adapted to oxygen (although ''Brettanomyces'' prefers the harsher environment availability of at least a little bit of wine 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>. Another [https://www.winesandvines.com/news/article/200000/New-Tools-to-Limit-Wine-Spoilage One study from Dr. Charles Edwards] found that one out a combination of keeping wine under 54°F (12.2°C) and alcohol at or above 14% resulted in a decline of the ''B. bruxellensis'' populations for up to 100 days for two strains that were tested . The study found that were isolated from soda could not ferment maltoseneither 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 only strains isolated from wine were 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 wine 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 beer/soda strains did notcells were inhibited at 200 IBU. The wine strains inhibited cells were also more resistant to sulfitesrecoverable in YPD media treated with catalase enzyme. In comparison, which are commonly used in the wine industry to prevent microbial contamination ''S. cerevisiae'' can be inhibited by 500 mg/L of iso-alpha acids <ref name=>[https://www.frontiersin.org/articles/10.3389/fmicb.2022.902110/full "Crauwels_2016Transcriptome 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>.

A The genetic survey diversity of ''Brettanomyces'' is particularly wide. For example, one study that analyzed the whole genomes of 1488 53 strains of ''B. bruxellensis'' found that the overall genetic diversity between different strains of ''B. bruxellensis'' from 29 countries, 5 continents, and 9 different fermentation niches was conducted in 2018 by Avramova et alhigher than strains of ''S. 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 groupcerevisiae'' (however, 1 kombucha group (most distantly related to the beer group)entire gene set, known as well as 1 tequila/ethanol group that has multiple ancestral lineages. This was expressed mostly in the ploidy level ''pangenome'', of each group (all the number genes among all of sets the strains of chromosomes''B. bruxellensis'' is much smaller than the entire gene set of ''S. cerevisiae'')<ref name="Gounot_2019" />. Some studies have indicated that strains of ''B. bruxellensis'' have adapted to specific environments. For example, with 2 one study found that strains of the ''B. bruxellensis'' isolated from wine groups, had 20 genes involved in the tequila groupmetabolism of carbon and nitrogen, and the whereas strains isolated from beer group containing more sets of chromosome pairs than the other groups (diploid vs triploid; this is thought to encourage adaption and hybridization)did not. Additionally, This indicated that ''B. bruxellensis'' strains living in wine have adapted to the triploid harsher environment of wine group was generally more tolerant of SO<supref name="smith_divol_2016">2</supref> than the diploid wine groups. The genetic differences between Another study found that one out of the fermentation substrates (beertwo strains tested that were isolated from soda could not ferment maltose, and only strains isolated from wine, etc.) were lower but still significant, able to grow in wine and this was explained by the frequent cross-over of equipment such as wine barrels being used for beer fermentation/soda strains did not. 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-The wine strainswere also more resistant to sulfites, suggesting that beer, kombucha, and tequila strains which are more localized genetically than wine strains and that humans probably helped commonly used in the wine strains travel across the globeindustry to prevent microbial contamination <ref name="Crauwels_2016" />. They also found that although The whole genome sequencing of one study reported spore-forming versions strain of ''B. bruxellensisnaardenensis'' (referred to as and lambic strains of ''Dekkera B. bruxellensis'')found that they are missing the genes associated with nitrate utilization, indicating that the genetic makeup assimilation of the analyzed strains determined their ability nitrates is not required 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 survive in 1960] has reported sporulation in ''Brettanomyces'' only on specific agar types with vitamins addedbeer, indicating that sporulation perhaps because of the abundance of nitrogen from other sources found in ''Brettanomyces'' is extremely rare) beer <ref name="Avramova_2018Tiukova_2019" />. See also [https:<ref name="colomer_2020_genome" //www.facebook.com/groups/MilkTheFunk/permalink/2022801681081369/ Richard Preiss's discussion of this study on MTF]>.

Sulfite and SO₂ inhibits the growth The addition of vitamins can have a positive impact on ''Brettanomyces''growth. For example, and is often used in the wine industry to prevent the growth of while ''Brettanomyces'' does not need riboflavin (vitamin B2) or thiamine (some wineries have identified small amounts vitamin B1) in order to grow, the presence of either or both of flavors from these two vitamins encourages ''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>) growth <ref>[http://onlinelibrary.wiley.com/doi/10.11111002/j.1745-4549.2012jib.00702.x385/abstract Removal full The influence of Brettanomyces Bruxellensis from Red Wine Using Membrane Filtrationthiamine and riboflavin on various spoilage microorganisms commonly found in beer. Umiker, DescenzoBarry Hucker, LeeMelinda Christophersen, and EdwardsFrank Vriesekoop. 04/24/20122017.]</ref>. HoweverOther vitamins such as p-aminobenzoic acid (PABA), folic acid (vitamin B9), it has been shown that wine nicotinic acid (vitamin B3), pantothenic acid (vitamin B5) are also not required for most strains of ''B. bruxellensisBrettanomyces'' could survive dosages of up to 1 mg/L grow. The presence of SO₂, and alcohol can increase the very high dosage dependence on vitamins for some strains of 2.1 mg/L was needed to kill ''Brettanomyces'' in wine <ref name="Agnolucci_2017" />to grow. This dosage of SO₂ is beyond legal limits For example, Myo-inositol (vitamin B8) and has negative effects on wine. The tolerance of SO₂ of thiamine (vitamin B1) were required by two strains of ''BrettanomycesB. bruxellensis'' found when grown in 10% ethanol but not in beer 0% ethanol. Biotin (vitamin B7) is lower than those the one exception, and it was found in wine strains. Some that the lack of biotin inhibited the growth of some strains of ''Candida pyralidaeB. bruxellensis''. Other studies contradict these previously mentioned findings, showing that thiamine was not required by the strains of ''Wickerhamomyces anomalusB. bruxellensis''tested, ''Kluyveromyces wickeramii'', ''Torulaspora delbrueckii'' and ''Pichia membranifaciens'' have been found to produce toxin that inhibits ''Brettanomyces''pyridoxine was required, and these toxins have been proposed as an alternative to SO₂ as a way to kill ''Brettanomyces'' (killer wine strains of ''Saccharomyces cerevisiae'' do biotin was not kill ''Brettanomyces''; see [[Saccharomyces#Killer_Wine_Yeast|Killer Wine Yeast]] for more information)required. [http://www.laboratoriosenosan.com/en/effectiveness-of-kaolin-silver-complex/ Kaolin silver complex (KAgC)] has been found These discrepancies between scientific studies are probably due to inhibit ''Brettanomyces'' and acetic acid bacteria in wine when used in legal dosagesthe genetic differences between the strains selected, the growth media chosen by the scientists, and has been proposed as a replacement for SO₂ or to minimize the use of SO₂ growth conditions <ref>[httpshttp://www.ncbimdpi.nlm.nih.govcom/2311-5637/2/pubmed3/29666535?dopt=Abstract Effect of kaolin silver complex on the control of populations 17 Use of Nutritional Requirements for Brettanomyces and acetic acid bacteria bruxellensis to Limit Infections in wineWine. Izquierdo-Cañas PM, López-Martín R, García-Romero E, González-Arenzana L, Mínguez-Sanz S, Chatonnet P, Palacios-García A, Puig-Pujol ANicolas H. von Cosmos and Charles G. 2018Edwards. DOI: 10.1007/s13197-018-3097-y2016.]</ref>.

The addition ====Sulfur Tolerance====  Sulfite and SO₂ inhibits the growth of ''Brettanomyces'', and is often used in the wine industry to prevent the growth of vitamins can ''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 positive impact 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 ''BrettanomycesB. 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 growtheventually) or resistant (no lagged growth, and achieved full growth) to 0.6 mg/L of molecular SO₂. 46 of the 52 resistant/tolerant strains were wine strains, thus demonstrating that wine strains of ''B. bruxellensis'' are generally more tolerant of SO₂ than strains of ''B. bruxellensis'' that are found in other types of beverages. It has been demonstrated that the wine strains have adapted to the conditions of winemakers adding SO₂ to wine <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, while ''Brettanomyces'' does 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 need riboflavin 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 (vitamin B2KAgC) ] 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 thiamine 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>. A study by Cibrario et al. (2019) looked at the genome of ''B. bruxellensis'' strains in bottles of wine going back as far as the year 1909 revealed that the SO₂ tolerant triploid strains only started appearing after the year 1990, which corresponds to when the wine industry started using SO₂ in most wine production (although it can also mean that the triploid strains are not as good at surviving in bottles of wine long-term compared to the diploid strains that have been isolated from much older bottles of wine; this will be determined to be the case or not in the future as bottles of wine from the 1990s continue to age). They also identified dozens of examples of wineries throughout France and Italy where the same strain of ''B. bruxellensis'' was found in multiple vintages of bottles of wine going back many decades, indicating that individual ''B. bruxellensis'' strains become long-term residents in wineries. Some identical strains have been found in different regions and even different continents, indicating that some strains have traveled not just due to traditional vectors such as insects or birds, but also probably due to human transportation such as wine bottle imports/exports or exchanges between industrial processes. This also indicates that while ''B. bruxellensis'' becomes rather sedentary and a constant resident in wineries, it can also adapt to the different winemaking conditions in different regions once it's been transported (different grapes, different climates, different fermentation temperatures, etc.), including adapting to improved modern hygienic practices such as higher SO₂ treatment. Overall, the results from this study suggest that ''Brettanomyces'' is able to adapt to living alongside certain human industries and has done so for at least a couple of centuries <ref name="Cibrario_2019">[https://www.biorxiv.org/content/10.1101/763441v1 Brettanomyces bruxellensis wine isolates show high geographical dispersal and long remanence in cellars. Alice Cibrario, Marta Avramova, Maria Dimopoulou, Maura Magani, Cécile Miot-Sertier, Albert Mas, Maria C. Portillo, Patricia Ballestra, View ORCID ProfileWarren Albertin, Isabelle Masneuf-Pomarede, Marguerite Dols-Lafargue. 2019. DOI: https://doi.org/10.1101/763441.]</ref>. The genetic differences between the fermentation substrates (vitamin B1beer, 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 order 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 growas ''Dekkera bruxellensis''), the presence genetic makeup of either the analyzed strains determined their ability to sporulate to be non-existent or both rare (only one study that we know of these two 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 encourages added, indicating that sporulation in ''Brettanomyces'' growth 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]. A study by [https://academic.oup.com/femsyr/advance-article-abstract/doi/10.1093/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" />. See also:* [http://onlinelibrarywww.foodsci.purdue.edu/research/labs/enology/Winemaking%20Calculations%20Spring%20Workshop%204_29_2011.pdf?fbclid=IwAR3WnDfo6k9hqfo4EvnWrp4GwFiB7qHoexifJBfLnIEIhLwRtC2Hm7qkUug Calculations for SO₂ additions] and [https://www.facebook.com/groups/592560317438853/?comment_tracking=%7B%22tn%22%3A%22R2%22%7D associated MTF discussion].* [[Quality_Assurance#VBNC_In_Yeast|Viable But Nonculturable Yeast.]] ====Biofilm====[[File:Chlamydospore Brett.JPG|thumb|First evidence of possible (unconfirmed <ref name="heit_lebleux">[https://www.facebook.com/groups/MilkTheFunk/permalink/3118617694833090/?comment_id=3120943487933844 Dr. Bryan Heit. Milk The Funk Facebook group thread on Lebleux et al. (2019) and chlamydospore in Brettanomyces. 12/11/2019.]</ref>) chlamydospore cell structures of ''B. bruxellensis'', found in a biofilm. Photo by [https://www.sciencedirect.com/science/article/abs/pii/S0168160519303952 Lebleux et al. (2019)] <ref name="Lebleux_2019">[https://www.wileysciencedirect.com/science/article/abs/pii/S0168160519303952 New advances on the Brettanomyces bruxellensis biofilm mode of life. Manon Lebleux, Hany Abdo, Christian Coelho, Louise Basmaciyan, Warren Albertin, Julie Maupeu, Julie Laurent, Chloé Roullier-Gall, Hervé Alexandre, Michèle Guilloux-Benatier, Stéphanie Weidmann, Sandrine Rousseaux. 2019. DOI: https://doi.org/10.1016/j.ijfoodmicro.2019.108464.]</ref>.]] ''Brettanomyces'' has the ability to form a [[Quality_Assurance#Biofilms|biofilm]]. Biofilm formation is a survival mechanism induced by stress whereby the cells adhere to non-living surfaces such as plastic and stainless steel <ref>[https://ives-technicalreviews.eu/article/view/4544 "Brettanomyces bruxellensis biofilms: a mode of life to withstand environmental stresses?" Sandrine Rousseaux, Manon Lebleux, Hany Abdo, Louise Basmacyian, Chloé Roullier-Gall, Hervé Alexandre, Stéphanie Weidmann. 2020. DOI: https://doi.org/10.20870/IVES-TR.2020.4544.]</ref>. After adhesion to the surface, the cells produce a protective layer of proteins and polysaccharides that help protect the organism from cleaning and sanitizing agents.  Joseph et al. (2007) tested 36 wine strains of ''B. bruxellensis'' for biofilm formation over a 10 day period. They found that just under half of the strains formed a biofilm, and about half of those formed considerable and consistent biofilms throughout the tests. Almost all strains tested (95%) adhered to a surface with 0.1% glucose within 6 hours of contact (the same conditions that get ''Saccharomyces cerevisiae'' to adhere to a surface; longer contact with surfaces and higher residual sugar could encourage ''Brettanomyces'' to adhere more readily to surfaces). A juice-based growth media in the range range of 2 - 4.5 pH was tested for biofilm formation and 3-4 for cell adhesion to a surface, and for most of the strains they formed stronger biofilms and adhered better in the higher pH growth media (4.5 pH being the highest tested). Under a pH of 3.5 significantly dropped biofilm formation and adherence, indicating that something about pH affects the cells ability to attach themselves. The researchers concluded that winemakers should keep wine in the lower end of the pH range (3.5). Six different types of cleaners were tested to see how well they removed the biofilms: keytones + surfactant detergent, quaternary ammonia + surfactant detergent, sodium hydroxide (caustic soda), sodium carbonate (soda ash), sodium hydroxide + surfactant (alkaline detergent), and chlorine (sanitizer, not a detergent). They found that only caustic soda was consistently efficient at removing the biofilm. The chlorine, while it did not remove the biofilm, still killed all of the ''Brettanomyces'' cells, and it was presumed that the other cleaners might have killed the ''Brettanomyces'', but that was not tested for. They also tested to see if cells that were adhered to a surface could be cleaned. Again, the caustic soda performed consistently the best, but the ammonia + surfactant cleaner and the quaternary ammonia + surfactant detergent also effectively removed adhered cells. The other cleaners varied in how well they removed adhered cells from a surface <ref>[https://www.researchgate.net/publication/235411588_Adhesion_and_biofilm_production_by_wine_isolates_of_Brettanomyces_bruxellensis Adhesion and biofilm production by wine isolates of Brettanomyces bruxellensis. C. M. Lucy Joseph, Gagandeep Renuka Kumar, Gagandeep Renuka Kumar, Edward Su, Linda F Bisson. 2006. American Journal of Enology and Viticulture 58(3):373-378.]</ref>.  Dimopoulou et al. (2019) studied ''Brettanomyces bruxellensis'' biofilms from each of the genetic branches of ''B. bruxellensis''. They found that for the wine strains biofilms formed more readily when grown in wine must rather than YPD media; however, the beer strains grew biofilms equally well in wine must and YPD media. The biofilms contained a large portion of saturated fatty acids and a smaller portion of monounsatured fatty acids. The amount of exopolysaccharide produced varied widely across the strains tested per cell population, with some wine strains producing little EPS (40 mg/L/OD), beer strains producing moderate amounts, and the other wine group producing a high amount (100 mg/L/OD). Additionally, the different strains displayed a varying degree of negative cell wall charges, with the beer and tequila strains being more negatively charged than wine strains, which could help them adhere to surfaces and form biofilm <ref>[https://europepmc.org/article/PPR/PPR73221?singleResult=true Dimopoulou M., Renault M., Dols-Lafargue M., Albertin-Leguay W., Herry J., Bellon-Fontaine M., Masneuf-Pomarede I. 2019. DOI: 10.10021101/jib579144.385]</full ref>. Lebleux et al. (2019) measured biofilm density for 12 strains across 5 of the genetic groups of ''B. bruxellensis''. All of the strains produced a biofilm when in contact with a surface (polystyrene and stainless steel, in the case of this study), and the thickness of the biofilm was proportional to the cell size of each strain. The influence biofilms contained filamentous cells that started from the base of thiamine the biofilm and riboflavin extended upward, indicating multiple layers. The biofilms also contained exopolysaccharides (EPS), but the makeup of the EPS was not analyzed and this was identified as a goal for further study. The average thickness was only 9.45 µm which is much thinner than other biofilm-forming yeast species (''Candida'' and a biofilm-producing strain of ''S. cerevisiae'' <ref>[https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-014-0305-4 Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on various spoilage microorganisms commonly growth phase. Bojsen, R., Regenberg, B. & Folkesson, A. BMC Microbiol 14, 305 (2014). DOI: 10.1186/s12866-014-0305-4.]</ref>). They found that one or two strains were less dense (contained fewer cells) than the average. A couple of the strains grew a biofilm a little slower than average. Two of the strains in beerbiofilm form were added to wine; each of the biofilms released cells into the wine, although one strain released more cells than the other. Introduction to the wine first led to cell death for some cells due to the harsh environment of the wine, but after several days the ''B. bruxellensis'' strains began to re-grow in the wine. Barry HuckerIt was observed that for one of the strains, the cells appeared larger than normal, Melinda Christophersenround, and had thicker cell walls, possibly forming what is known as [https://en.wikipedia.org/wiki/Chlamydospore chlamydospore cell structures]. It was not confirmed in the study whether these cells were actually chlamydospores, Frank Vriesekoopand their structure could be due to relatively insignificant reasons <ref name="heit_lebleux" />. 2017Chlamydospore cell structures are known to help certain species of non-yeast fungi survive harsh environments; however, it has not yet been established that yeast with chlamydospore cell structures helps them survive harsh conditions, and this was also identified in the study as an area for further research <ref name="Lebleux_2019" />. See also:* [[Quality Assurance]]* [[Pellicle]] ====UV Light====There is some evidence that ''Brettanomyces'' can be sensitive to high levels of light. [https://www.frontiersin.org/articles/10.3389/fmicb.2021.747868/full Catrileo et al. (2021)] showed that under laboratory conditions, ''Brettanomyces bruxellensis'' was not able to grow when exposed to a 2500 lux and 4000 lux light source. For reference, the lux of indirect daylight is around 10,000 - 25,000 and the lux of office lighting is usually between 350 and 500 <ref>[https://en.wikipedia.org/wiki/Lux "Lux". Wikipedia. Retrieved 02/20/2022.]</ref>. However, when p-coumaric acid, a phenolic precursor that is present in plants and fruits (including malted barley and wheat), is present, certain genes are expressed during the growth of ''B. bruxellensis'' that allow it to adapt to the high light exposure conditions. While this study does not show at what level light begins to affect ''B. bruxellensis'' (the lowest light intensity that they tested was 2500 lux), [https://journals.asm.org/doi/abs/10.1128/jb.133.2.692-698.1978 Woodward et al. (1978)] demonstrated that ''Saccharomyces cerevisiae'' growth is unaffected by light until about 1,250 lux, at which point it begins to inhibit growth and the transfer of nutrients across the cell membrane <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2021.747868/full Catrileo D, Moreira S, Ganga MA and Godoy L (2021) Effect of Light and p-Coumaric Acid on the Growth and Expression of Genes Related to Oxidative Stress in Brettanomyces bruxellensis LAMAP2480. Front. Microbiol. 12:747868. doi: 10.3389/fmicb.2021.747868.]</ref><ref>[https://journals.asm.org/doi/abs/10.1128/jb.133.2.692-698.1978 J R Woodward, V P Cirillo, L N Edmunds, Jr. Light effects in yeast: inhibition by visible light of growth and transport in Saccharomyces cerevisiae grown at low temperatures. ASM Journals. Journal of Bacteriology. Vol. 133, No. 2. 1978. https://doi.org/10.1128/jb.133.2.692-698.1978.]</ref>. As a follow up question within Milk The Funk group on Facebook regarding if lower levels of light could impact ''Brettanomyces'' growth, Richard Preiss of Escarpment Labs performed an in-house experiment to grow ''Brettanomyces'' in the presence of standard fluorescent lights and reported finding no impact of the lights on ''Brettanomyces'' growth <ref>[https://www.facebook.com/groups/MilkTheFunk/posts/5523998620961640/?comment_id=558987711104045 Richard Preiss. Milk The Funk Facebook group post on impact of light on ''Brettanomyces'' growth. 03/07/2022.]</ref>.

[[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>. The Custers effect is abolished under anaerobic conditions when [https://en.wikipedia.org/wiki/Nitrate nitrate] is available. Under conditions where there is no oxygen, as long as nitrates are available, it has been shown that ''Brettanomyces'' can produce ethanol just as capably as ''S. cerevisiae'' <ref>[https://link.springer.com/article/10.1007%2Fs10295-012-1229-3 Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products. Silvia Galafassi, Claudia Capusoni, Md Moktaduzzaman, Concetta Compagno. 2013.]</ref><ref>[https://link.springer.com/article/10.1007/s10295-018-2118-1 Nitrate boosts anaerobic ethanol production in an acetate-dependent manner in the yeast Dekkera bruxellensis. Irina Charlot Peña-Moreno, Denise Castro Parente, Jackeline Maria da Silva, Allyson Andrade Mendonça, Lino Angel Valcarcel Rojas, Marcos Antonio de Morais Junior, Will de Barros Pita. 2018.]</ref>. Brewers who prefer the character of ''Brettanomyces'' in their beer can take advantage of this ability by limiting oxygen and providing a food source for ''Brettanomyces'' (aka beer or wort). See [[Brettanomyces#Nitrogen_Metabolism|Nitrogen Metabolism]] below.

''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'' an achieve in beers such as lambic and American sour beers <ref name="yakobson_introduction"></ref><ref name="smith_divol_2016"></ref>. Alpha-glucosidases are the enzymes that allow them to break down maltose, turanose, melezitose, and trehalose, as well as dextrins such as maltotetraose and maltopentaose. These enzymes work by cleaving off glucose that can be directly consumed by the cell, leaving a shorter chain sugar behind which is then further broken down. In the case of extracellular alpha-glucosidase activity, this breakdown of complex sugars occurs outside of the cell and may benefit other microorganisms if present such as lactic acid bacteria. These dextrins are left over after a normal ''Saccharomyces'' fermentation <ref name="Steensels"></ref>. Some other polysaccharides can be fermented by ''Brettanomyces'', including starch, laminarin, and pectin <ref name="Crauwels1"></ref>. The more complex the starch or sugar, the slower it is hydrolyzed by the alpha-glucosidase enzymes. The optimal pH for the alpha-glucosidase enzyme produced by one strain of ''B. bruxellensis'' was 6 and at a temperature of 39-40°C (102-104°F), and its activity was greatly reduced below a pH of 4.5 and above 8 (although citric acid was used as a buffer, and its effects on the enzyme was not compared to other acids), which might contribute to slower ''Brettanomyces'' fermentation in acidic beers <ref name="Kumara_1993"/>. A survey of 84 strains from several species of ''Brettanomyces'' showed that there is a wide variability in the ability of different strains to ferment maltose, with some strains not being able to ferment it at all and others fermenting it very slowly, suggesting that alpha-glucosidase is not functional or poor in some strains. Additionally, when maltose is present instead of just glucose, the researchers saw an increased lag during the growth phase <ref name="colomer_2020_genome" />.  ''B. bruxellensis'' and ''B. nanus'' can also produce oligo-1,6-glucosidase, which hydrolyze the alpha-1,6 linkages in starch and glycogen to produce oligosaccharides, and then further break down these oligosaccharides to produce sugars with alpha-1,4 linkages (for example, maltose, in the case of starches from malted barley <ref>[httphttps://aemwww.ncbi.nlm.asmnih.orggov/contentbooks/59NBK22396/8Complex Carbohydrates Are Formed by Linkage of Monosaccharides. Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. 2002.]</2352ref>).short Localization and Characterization These alpha-1,4 linkages (maltose) are then further broken down by the maltose enzyme by strains of α-Glucosidase Activity ''Brettanomyces'' or other present microbes that produce this enzyme <ref name="Crauwels_2014" />. Unlike for some domesticated diastatic ''S. cerevisiae'' strains, this ability for ''Brettanomyces'' to break down starches has occurred in Brettanomyces lambicusthe wild without domestication <ref name="roach_2019" /><ref>"Amylases". H. MTaniguchi, Y. Honnda, in Encyclopedia of Microbiology (Third Edition), 2009. C]</ref><ref>[https://www. Shantha Kumara, Sfacebook. De Cort and Hcom/groups/MilkTheFunk/permalink/3049768678384659/ Ben Sykes. Verachtert Milk The Funk Facebook post on ''Brettanomyces'' evolution to ferment dextrins. 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 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 fermented were generally better at fermenting cellobiose while than strains isolated from beer did not 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/1285391944822350permalink/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>. Fermentation At 20°C (68°F) fermentation rate is about half as slow at 20°C (68°F). ''Brettanomyces'' will still grow at temperatures as low as 15°C (and maybe lower than59°F) 15°C with about a third of strains being able to grow as low as 10°C (59°F50°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, 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 deathfor most strains of ''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 isolated from beer at 15°C (59°F). However, at 22.5°C (72.5°F) most of the beer strains started to utilize cellobiose after a few days of incubation (they preferred other food sources such as glucose and maltose), indicating that temperature plays a role in whether ''Brettanomyces'' can ferment certain sugars <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 are ''B. bruxellensis'' strains that are isolated from beer generally unable to break down glycosides, but they are probably also unable to utilize the cellobiose in wooden barrels as a food source (although higher temperature might allow some beer strains to start fermenting cellobiose).

Other than sugars, nitrogen in the form of amino acids is an essential nutrient for yeast and generally occurs . One significant source for nitrogen in wort in the form of amino acids is boiling hops and dry hopping <ref>Principles of Brewing Science, A Study of Serious Brewing Issues. George Fix. Brewers Publications. 1999. Pg 40.</ref><ref>[https://www.researchgate.net/publication/286126882_Transfer_of_nitrate_and_various_pesticides_into_beer_during_dry_hopping Transfer of nitrate and various pesticides into beer during dry hopping. M. Kippenberger, S. Hanke, M. Biendl, G. Stettner and A. Lagemann. 2014.]</ref>. ''Brettanomyces'' can survive in environments that are very low in nitrogen, with one report being as low as 6 mg N/L of yeast assimilable nitrogen (YAN) which is less than most finished wines contain <ref>[https://www.ncbi.nlm.nih.gov/m/pubmed/25475334/ Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis. Childs BC, Bohlscheid JC, Edwards CG. 2015.]</ref>. While nitrogen usage for ''S. cerevisiae'' is well understood, the general utilization of nitrogen by ''Brettanomyces'' and its preferred sources for nitrogen under the stressful conditions of fully fermented beer and wine are not yet well known. However, it is known that ''Brettanomyces'' can use a wide range of sources for nitrogen, and its requirements for nitrogen as a nutrient are extremely low when oxygen is available. When oxygen is not present, nitrogen is required for the survival and growth of ''Brettanomyces''. Sources Preferred sources of nitrogen include the amino acids glutamine (aerobically and anaerobically), glutamate and aspartate (anaerobically) <ref>[https://www.ncbi.nlm.nih.gov/pubmed/29065215 On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes. Parente DC, Cajueiro DBB, Moreno ICP, Leite FCB, De Barros Pita W, De Morais MA Jr. 2018. DOI: 10.1002/yea.3290.]</ref>, as well as possible secondary sources such as lysine, histidine, arginine, asparagine, aspartic cid, glutamic acid, and alanine <ref name="smith_divol_2016" />. Some strains of ''Brettanomyces'' can metabolize other nitrogen sources, such as the amino acids proline and arginine <ref name="Crauwels1"></ref>. Ammonium nitrates may also be utilized by some strains of ''B. bruxellensis''. Although studies have been contradictory and some have not documented whether conditions were aerobic or anaerobic (these contradictions might also be due to strain differences between the ''B. bruxellensis'' strains that were used in different studies), it appears as though some strains of ''B. bruxellensis'' might be able to take advantage of trace amount of amino acids that ''S. cerevisiae'' does not use during fermentation, and nitrates and nitrites that ''S. cerevisiae'' is not able to consume, as well as amino acids from yeast autolysis (proline, leucine, tryptophan, and gamma aminobutyric acid) <ref name="smith_divol_2016"></ref>. Other compounds from ''Saccharomyces'' autolysis may also be used by ''Brettanomyces'', such as glucose, fatty acids, nucleotides, polysaccharides, polypeptides, and other proteins <ref>Private correspondence with Richard Preiss by Dan Pixley. 08/23/2016.</ref><ref>[http://oeno-one.eu/article/view/1701 Influence of yeast autolysis after alcoholic fermentation on the development of Brettanomyces/Dekkera in wine. Michèle Guilloux-Benatier, D. Chassagne, Hervé Alexandre, Claudine Charpentier, Michel Feuillat. 2001.]</ref>. The role that oxygen plays in the ability of ''B. bruxellensis'' to uptake nitrogen from various sources might be an important one, and something that should be examined in science going forward <ref name="smith_divol_2016"></ref>.

''Secondary metabolites'' are compounds that are not essential to the life of an organism <ref>[http://en.wikipedia.org/wiki/Secondary_metabolite Wikipedia. Secondary Metabolite. Retrieved 6/2/2015.]</ref>. ''Brettanomyces'' will use a range of secondary metabolites to produce many of the fruity and funky esters, phenols, and acids that this genus of yeast has become known for. ''Brettanomyces'' has also been observed anecdotally to produce thin beer when fermented on its own, and this has at least partially been attributed to the lack of glycerol production by ''Brettanomyces''. The lack of glycerol production has been attributed to a genetic predisposal to prefer pyruvate production over glycerol production during fermentation, and it has been speculated that this gives ''Brettanomyces'' an adaptive advantage <ref>[http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0155140 Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts. Yi-Cheng Guo, Lin Zhang, Shao-Xing Dai, Wen-Xing Li, Jun-Juan Zheng, Gong-Hua Li, Jing-Fei Huang. 2016.]</ref><ref name="yakobson_introduction"></ref>. The major secondary metabolites of ''B. bruxellensis'' fermentation have been identified in one study as the ethyl phenols (4EP and 4EG), the alcohols isoamyl alcohol, 2-methyl-butanol, 2-ethylhexanol, phenethyl alcohol, and an ester ethyl 2-methyl butyrate. Many other compounds are considered minor secondary metabolites and are produced in varying degrees or not at all based on the strain of ''Brettanomyces'', but may still be produced in high enough concentrations to contribute to the flavor and/or aroma in beers fermented with ''Brettanomyces''. The types and amounts of flavor compounds produced by ''Brettanomyces'' cover a wide spectrum, and many factors such as species/strain, amino acid precursors, the presence of oxygen, and other nutrients, play a large role in the production of these compounds. In one study on ''Brettanomyces'' in wine, some strains rated as being perceived positively if the strains metabolized certain compounds slower and produced other compounds slower, indicating that the age of the fermented beverage also plays a large role in how beverages fermented with ''Brettanomyces'' are perceived <ref name="Lucy_2015" />. Some strains of ''B. bruxellensis '' also produce the amines cadaverine, hexylamine, phenylethylamine, putrescine and spermidine, under wine-model conditions <ref name="Agnolucci_2017" />.

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" />.

Pitching rate of ''Brettanomyces'' may have a slight effect on ester production levels, but the differences caused by pitching rate probably do not have a significant impact on the sensory character of the beer <ref name="MTF_Brett_Secondary"></ref>. ''Brettanomyces'' produces higher levels of esters when fermented without competition from ''S. cerevisiae'', and this correlates with higher ''Brettanomyces'' cell growth when not in competition with ''S. cerevisiae'' (see [[100%25_Brettanomyces_Fermentation#Are_100.25_Brett_Beers_Really_Cleaner.3F|100% ''Brettanomyces'' Fermentation]]) <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/permalink/1407620509266159/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. The aromatic amino acids phenylalanine, tryptophan, and tyrosine have been associated with higher ester formation <ref name="Lucy_2015" />. High levels of bicarbonate can affect the ester production of ''B. bruxellensis'', as well as the production of acids and phenols. One study reported that levels of 100 mg/l produced significantly higher ethyl acetate, but there was less of an effect on other esters. High amounts of bicarbonate over 100 mg/l in the 100% ''B. bruxellensis'' fermentations produced significantly lower amounts organic acids (hexanoic, octanoic, and decanoic acid) and lower amounts of vinyl phenols <ref name="Thompson-Witrick_2022" />. See also The Brü Lab Podcast with [http://thebrulab.libsyn.com/episode-087-impact-bicarbonates-have-on-brettanomyces-fermentations-w-dr-katherine-thompson-witrick Dr. Thompson-Witrick].

[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>. While both Some strains of ''Oenococcus oeni'' and ''Lactobacillus'', as well as some strains of yeast such as ''SaccharomycesPichia'' (only 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 "phenolic off flavor positive/POF+" strains) and ''Brettanomyces'' strains were the same as wine that did not have varying capabilities based on strain of converting hydroxycinnamic acids an increase in HCA levels (the precursors in wine that lead to their vinyl derivatives HCA's are different than they are in beer) <ref name="Lentz">[http://www.mdpiajevonline.comorg/content/2304-8158early/42018/405/58102/htm Analysis ajev.2018.17092 Influence of Growth Inhibition Oenococcus oeni and Metabolism of Hydroxycinnamic Acids by Brewing Brettanomyces bruxellensis on Wine Microbial Taxonomic and Spoilage Strains of Brettanomyces YeastFunctional Potential Profile. Michael Lentz and Chad HarrisMarie 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. 2015DOI: 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''Brettanomyces'' s can then be released from these esters. This formation of esters is also able 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 reduce these vinyl phenol derivatives to ethyl phenol derivativesEthylphenols 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>. Phenolic acid decarboxylase (PAD) 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 enzyme release of HCA's from barley (although this lab technique uses a lower pH then that converts the HCAs into vinyl phenolsof sour beer). Vinyl reductase In addition, it has been demonstrated that spent yeast (VA''S. cerevisiae'' collected after beer fermentation) is the enzyme that reduces vinyl phenols to ethyl contains a small fraction of phenols and polyphenols absorbed from wort during fermentation <refname="Cortese_2020">[https://www.facebooksciencedirect.com/groupsscience/MilkTheFunkarticle/1632316743463200pii/ Analysis S0021967319310295 Quantification of phenolic acid decarboxylase enzyme from the wine spoilage yeast Brettanomyces bruxellensis (poster)compounds in different types of crafts beers, worts, starting and spent ingredients by liquid chromatography-tandem mass spectrometry. Mike LentzManuela Cortese, Maria Rosa Gigliobianco, Dolores Vargas Peregrina, Jamie LynchGianni Sagratini, Pricilla WaltersRoberta Censi, Rachel LiceaPiera Di Martino. Journal of Chromatography A; Volume 1612, Henok Daniel8 February 2020, Kimberly Pereira460622. 2017DOI: https://doi.org/10.1016/j.chroma.2019.460622.]</ref>. It is therefore conceivable that HCA levels could increase in sour beer over time.

These vinyl derivatives have similar tastes to the ethyl derivatives but have lower While both ''Saccharomyces'' (only by "phenolic off flavor thresholds. Levels of all compounds produced vary depending on species positive/POF+" strains) and strain of ''Brettanomyces''. Although the production strains have varying capabilities based on strain of ethyl phenols has been identified converting hydroxycinnamic acids to occur higher in substrates with more available sugars, and this has also correlated with higher growth their vinyl derivatives <ref name="Barata_2008">[httphttps://onlinelibrarylink.wileyspringer.com/doiarticle/10.11111007/js10482-016-0793-3 González, C.1567-1364, Godoy, L.2008& Ganga, M.00415A.x/full The effect Identification of sugar concentration and temperature on growth and volatile phenol production by Dekkera a second PAD1 in Brettanomyces bruxellensis in wineLAMAP2480. André BarataAntonie van Leeuwenhoek 110, Daniela Pagliara, Tiziana Piccininno, Francesco Tarantino, Wilma Ciardulli, Manuel Malfeito-Ferreira, Virgílio Loureiro291–296 (2017). 2008https://doi. DOI: org/10.11111007/j.1567s10482-016-0793-1364.2008.004153.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_SecondaryLentz">[http://www.milkthefunkmdpi.com/wiki2304-8158/4/4/581/Brettanomyces_secondary_fermentation_experiment htm Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces secondary fermentation experimentYeast. Milk The Funk WikiMichael Lentz and Chad Harris. Lance Shaner and Richard Preiss2015.]</ref><ref>[https://www.biorxiv.org/content/10.1101/2024.04.16. 586637v1 Retrieved Characterization of Brettanomyces bruxellensis phenolic acid decarboxylase enzyme expressed in E. coli. Michael R. Lentz. bioRxiv 2024.04.16.586637; doi: https://doi.org/2110.1101/20162024.04.16.586637.]</ref>. Additionally, Curtin et al''Brettanomyces'' is also able to reduce these vinyl phenol derivatives to ethyl phenol derivatives. Phenolic acid decarboxylase (2013PAD) showed that while both cell growth and attenuation was inhibited in anaerobic conditions in wine, phenol production was not (in fact, is the phenol production was inhibited by aerobic conditions). They also showed enzyme that each of converts the three strains of ''BHCAs into vinyl phenols. bruxellensis'' tested all produced Vinyl reductase (VA) is the same amount of enzyme that reduces vinyl phenols to ethyl phenols, while other flavor compounds such as esters were produced at different levels by each of the strains <ref>[https://www.ncbifacebook.nlm.nih.govcom/groups/MilkTheFunk/permalink/pubmed1632316743463200/24010603 Impact Analysis of Australian Dekkera phenolic acid decarboxylase enzyme from the wine spoilage yeast Brettanomyces bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma(poster). Curtin CDMike Lentz, Langhans GJamie Lynch, Henschke PAPricilla Walters, Grbin PRRachel Licea, Henok Daniel, Kimberly Pereira. 20132017.]</ref>. Perhaps growth itself is not as much Phenol production has been observed to occur shortly after inoculation of ''Brettanomyces'' and has been hypothesized to play a factor large role in producing phenols, but having sugars available for metabolism is. This contradicts replenishing NAD⁺ to alleviate the somewhat popular belief that under-pitching initial lag growth phase in ''Brettanomyces'' produces more <ref name="funkyTyrawa_Masters" flavors>[https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation. AdditionallyUniversity of Guelph, perhaps some Masters Thesis. Department of Molecular and Cellular Biology. 2020.]</ref>. While almost all strains are perceived as of ''Brettanomyces'' produce ethyl phenols, one strain of ''Brettanomyces anomalus'' has been found that has lost the genetic capability to produce phenols <ref name="funkiercolomer_2020_genome" than others due to less ester production and more fatty acid production (isobutyric acid, for example), rather than more phenol production/>.

Another study 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>[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 Tyrawa et alDekkera 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. found x]</ref>, some data supports that fermentation temperature also did pitching rate does not have a significant 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 9 fact, the phenol production was inhibited by aerobic conditions). They also showed that each of the three strains of ''B. bruxellensis''. Given tested all produced the same wort 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 compositionand 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'' produced similar levels <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 phenols at both 15°C Brettanomyces Yeast Strains in Primary and 22Secondary Beer Fermentations.5°C]</ref>. The ester production was affected by this temperature difference Perhaps growth itself is not as much of a factor in some strains producing phenols, but not others (see [[Brettanomyces#Ester_Production|Esters]] above)having sugars available for metabolism is. Assuming This contradicts the somewhat popular belief that phenols contribute the under-pitching ''Brettanomyces'' produces more "funky" flavor characteristicsflavors. Additionally, this suggests that perhaps a lower balance of esters to phenols produces a more some strains are perceived as "funkyfunkier" tasting beer than others due to less ester production and more so fatty acid production (isobutyric acid, for example), rather 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" />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>.  It has been demonstrated in wine that some phenols can be masked by other flavor compounds, especially lower levels of phenols. Schmuaker et al. (2018) showed that wines that were spiked with 4EG, 4EP, and isobutyric acid were preferred more when additionally spiked with whiskey lactone (oaky flavor). The oaky flavor at least partially masked the perception of phenols on the palate <ref>[https://www.sciencedirect.com/science/article/pii/S0963996918307567 Influence of wine composition on consumer perception and acceptance of Brettanomyces metabolites using temporal check-all-that-apply methodology. Megan R. Schumaker, Charles Diako, John C. Castura, Charles G. Edwards, Carolyn F. Ross. 2018. DOI: https://doi.org/10.1016/j.foodres.2018.09.034.]</ref>. It has been hypothesized by some members of Milk The Funk that higher esters could also mask the perception of phenols in beer (see [[100%25_Brettanomyces_Fermentation#Questioning_Conventional_Wisdom|100% ''Brettanomyces'' fermentation]]).

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>.

| 4-Ethylphenol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Barnyard, Horsey, Spicy, Smoky, Medicinal, Band-Aid <ref>[http://www.aroxa.com/wine/wine-flavour-standard/4-ethyl-phenol/ Aroxa Website. 4-Ethyl Phenol. Retrieved 08/19/2015.]</ref>) || Ethyl phenol || 4-vinylphenol || 0.3 ppm (odor; in beer) <ref name="Maarse">[https://books.google.com/books?id=_OvXjhLUz-oC&pg=PA513&lpg=PA513&dq=4-ethylphenol+odor+threshold&source=bl&ots=fzhA9yvvrJ&sig=K0QykyRqj9TnezG1Mih4gLru1ZE&hl=en&sa=X&ved=0CC4Q6AEwAmoVChMI77DfhNi1xwIV0jqICh1zVgQ8#v=onepage&q=4-ethylphenol%20odor%20threshold&f=false Volatile Compounds in Foods and Beverages. Henk Maarse. CRC Press, Mar 29, 1991. Pg 514, 515.]</ref> || C₈H₁₀O <ref name="pubchem_4EP">[http://pubchem.ncbi.nlm.nih.gov/compound/4-ethylphenol PubChem Website. 4-Ethylphenol. Retrieved 08/19/2015.]</ref> || Also known as 1-Ethyl-4-hydroxybenzene and P-Ethylphenol <ref name="pubchem_4EP"></ref>. Identified as a major product of ''B. bruxellensis'' <ref name="Lucy_2015" />. Richard Preiss of [[Escarpment Laboratories]] describes pure 4EP as the following, "barnyardy with a slight solvent edge at low concentrations, and full on hospital antiseptic/bandaid/barnyard at high concentrations. Really quite complex, but maybe not in a good way." <ref name="Preiss_4EP_4EG">[https://www.facebook.com/groups/MilkTheFunk/permalink/2141048572590012/?comment_id=2141122099249326&reply_comment_id=2141606422534227&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss. Milk The Funk Facebook group thread on the flavor of 4EP and 4EG. 06/22/2018.]</ref>

| 4-Ethylguaiacol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Smokey, Spicy, Clove <ref>[http://www.aroxa.com/discovery/discovery-standard/4-ethyl-guaiacol Aroxa Website. 4-Ethyl guaiacol. Retrieved 08/19/2015.]</ref>) || Ethyl phenol || 4-vinylguaiacol || 0.13 ppm (odor; in beer) <ref name="Maarse"></ref> || C₉H₁₂O₂ <ref name="pubchem_4EG">[http://pubchem.ncbi.nlm.nih.gov/compound/62465 PubChem Website. 4-Ethyl-2-methoxyphenol. Retrieved 08/19/2015.]</ref> || Also known as 4-Ethyl-2-methoxyphenol <ref name="pubchem_4EG"></ref>. Identified as a major product of ''B. bruxellensis'' <ref name="Lucy_2015" />.Richard Preiss of [[Escarpment Laboratories]] describes pure 4EP as the following, "4EG is a lot more pleasant (than 4EP), very light woodsmoke at low concentrations and heavier smoke and vanilla-like (actually "tonka bean" is closer but not as universally understood) at high concentrations." <ref name="Preiss_4EP_4EG" />

In the presence of oxygen, ''Brettanomyces'' strains species produce acetic acid as a byproduct of glucose respiratory 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 fermentationand 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 [[Secondary metabolitesBrettanomyces#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 <ref name="mello_funkweapon2">[https://www.facebook.com/groups/MilkTheFunk/permalink/1955043657857172/?comment_id=1955050584523146&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Jeff Mello. Milk THe Funk thread on Funk Weapon species. 01/15/2018.]</ref> || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || Funk Weapon #1 BB0034A || This rare, and commercially unavailable yeast isolate, produces pungent horse blanket and fresh leather aromas. Perfect for breaking out the funk in farmhouse-style beers. This is the first release in the Dusty Bottoms Collection’s ongoing Funk Weapon Series of unique, rare Brettanomyces and Brett-like wild yeast cultures. Single isolate <ref name="mello_funkweapon">[https://www.facebook.com/groups/MilkTheFunk/permalink/1935201836508021/?comment_id=1935238029837735&reply_comment_id=1935262719835266&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Jeff Mello. Milk The Funk post on Funk Weapon. 12/29/2017.]</ref>. || West Flanders, Belgium brewery specializing in funky, sour, mixed-fermentation beers.

| Bruxellensis <ref name="mello_funkweapon2" /> || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || Funk Weapon #2 BB0035C || This rare, and commercially unavailable yeast isolate is perfect for 100% Brettanomyces fermentations, especially Brett IPAs. Amplifies citrus and tropical fruit-forward hop flavors and aromas into a punchy ripeness. Great for maintaining the nuance of hops in beer with greater keeping qualities than a Brewer’s Yeast fermentation. You can’t make every style of beer with one or two strains of brewer’s yeast, so why would you only use only one or two strains for your funky beers? This is the second release in the Dusty Bottoms Collection’s ongoing Funk Weapon Series of unique, rare Brettanomyces and Brett-like wild yeast cultures. Single isolate <ref name="mello_funkweapon" />. || Family-owned brewery producing Gueuze in West Flanders, Belgium.

| Bruxellensis <ref name="mello_funkweapon2" /> || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || Funk Weapon #3 BB0022 || Funk Weapon #3 is a versatile culture that creates wildly different flavor and aroma profiles depending on the age of fermentation. Young fermentations produce mild musty funk and ripe tropical fruit, while older and bottle conditioned ferments show off unique flavors and aromas of strawberry, cherry and tropical candy. This commercially unavailable yeast isolate is ideal for 100% Brettanomyces fermentations or as a secondary strain along with a phenolic Brewer’s Yeast culture. Single isolate <ref name="mello_funkweapon" />. || Dry-hopped, Unblended Lambic Produced by a Traditional Lambic Brewery in Brussels, Belgium.

| Bruxellensis||''Dekkera bruxellensis''||''B. Brettanomyces bruxellensis''||Brettanomyces bruxellensis||Medium intensity Brett character. Classic strain used in secondary fermentation for Belgian style beers and lambics. || Same Thought to be the same as White LabsWLP648; 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.'''

| Claussenii||''Dekkera anomala''||''B. Brettanomyces anomalus''||Brettanomyces clausenii||Low intensity Brett character. ||Same Thought to be the same as White Labs. '''Commercial pitches only.'''

| CMY1||''Dekkera bruxellensis''||''B. Brettanomyces bruxellensis''||bsi|| ||Chad Yakobson's mutation of BSI Drie. '''Commercial pitches only.'''

| Drie||''Dekkera bruxellensis''||''B. Brettanomyces bruxellensis''||Brettanomyces bruxellensis var. Drei||Highly aromatic brett strainof tropical fruit come with aging. Works well with citrus and fruity hops. Sourness takes extensive aging to produce||Isolate from Drie Fonteinen; Pro-Brewers only. Recommended fermentation levels to get the most character out of Drie include 75-77°F <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1231171843577694/?comment_id=1231195766908635&reply_comment_id=1231197923575086&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Mark Horsley and Ed Coffey on MTF. 02/11/2016.]</ref>. '''Commercial pitches only.'''

| Lambicus||''Dekkera bruxellensis''||''B. Brettanomyces bruxellensis''||Brettanomyces lambicus||High intensity Brett character. Know to produce the “horsey” aroma characteristic of Brettanomyces yeast. Classic strain used in secondary fermentation for Belgian style beers and lambics. Same as White Labs. || '''Commercial pitches only.'''|-|} ===[[Community Cultures Yeast Lab]]==={| class="wikitable sortable"|-! Common Name !! Species Name !! Synonym Name !! Lab/Package !! Flavor/Aroma !! Source Note|-| Claussenii || ''Dekkera anomala'' || ''Brettanomyces anomalus'' || Brettanomyces claussenii || A little less "Bretty" and a little more fruity. Flocculation: Low. Alcohol Tolerance: Medium-High (8-12%). Fermentation Temperature: 85F. |||-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || Brettanomyces bruxellensis || For secondary fermentation and Belgians, with classic "Bretty" characters. Flocculation: Low. Alcohol Tolerance: Medium-High (8-12%). Fermentation Temperature: 85F. ||

| Bruxellensis || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || CCYL411 Brettanomyces bruxellensis || Exhibits medium intensity Brett character <ref name="craft_cultures" />. || '''Commercial pitches only.'''

| Claussenii || ''Dekkera anomala'' || ''B. Brettanomyces anomalus'' || CCYL412 Brettanomyces claussenii || Low intensity Brett character. The Brett flavors produced are more subtle than CCYL410. More aroma than flavor contribution. Fruity, pineapple like aroma <ref name="craft_cultures" />. || 75-85% attenuation, low flocculation, 8-12% alcohol tolerance, 67-74°F fermentation temperature <ref name="craft_cultures" />. '''Commercial pitches only.'''

| Lambicus || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || CCYL413 Brettanomyces lambicus || High intensity Brett character. Defines the "Brett character": Smoky and spicy flavors. This strain is often found in Lambic, Flanders, and sour brown style beers <ref name="craft_cultures" />. || '''Commercial pitches only.'''

| FarmhouseAnomala||?''Dekkera anomala''||?''Brettanomyces anomalus''||ECY-03b04||Fruity This species displays strong esters of juicy-fruit gum, moderate funk and funky profile with some gradual acidity gradually increasing over time. Aeration has more of a muted effect. Identified as ''Saccharomyces boulardii'' A super-attenuator, up to 100% <refname="ecy_website">[httpshttp://www.facebookeastcoastyeast.com/groupswild-stuff.html "Wild Yeast /MilkTheFunkBrettanomyces /permalinkLactic Bacteria". East Coast Yeast website. Retrieved 04/97254327944055327/?comment_id=972571192771095&offset=50&total_comments=73&comment_tracking=%7B%22tn%22%3A%22R9%22%7D May not be Brett as per Lance Shaner on MTF2018.]</ref>; no longer available as a single strain, but is included in ECY03 Farmhouse Blend <ref name="ecy_website" />. ||Isolate from Fantome. beer - Adelaide, Australia

| AnomalaBruxellensis||''Dekkera anomalabruxellensis''||''B. anomalusBrettanomyces bruxellensis''||ECY-0405|| This species displays strong esters of juicy-funky with barnyard notes accompanied by some fruit gum, moderate funk and gradual acidity over time. A super-attenuator, up to 100% May no longer be available <ref name="ecy_website"/>[http://www.eastcoastyeast.com/wild-stuff.html "Wild Yeast / Brettanomyces / Lactic Bacteria". East Coast Yeast website||isolated from Belgian stout. Retrieved 04/27/2018.]</ref>||beer - Adelaide, Australia

| BruxellensisNanus||''Dekkera bruxellensisBrettanomyces nanus'' ||''B. bruxellensisBrettanomyces nanus''||ECY-0516||funky This species of ''Brettanomyces'' reveals a phenolic, spicy, saison-like profile with barnyard notes accompanied by some fruitnone to low esters. May no longer Some earthiness and tartness may develop over time. Slow to attenuate fully alone, may be available <ref name="ecy_website" />best in fermenting with other yeast. ||isolated from Belgian stout. bottled beer - Kalmar, Sweden

| NanusCustersianus||''B. nanusBrettanomyces custersianus'' ||''B. nanusBrettanomyces custersianus''||ECY-1619||This species of ''Brettanomyces'' reveals a phenolic, spicy, saison-like profile with none to low esters. Some earthiness Light fruit and tartness may develop over timehay. Slow to attenuate fully alone, may May no longer be best in fermenting with other yeastavailable <ref name="ecy_website" />. ||bottled Bantu beer - Kalmarbrewery, SwedenSouth Africa

| CustersianusNaardenensis||''Brettanomyces custersianusnaardenensis'' ||''B. custersianusBrettanomyces naardenensis''||ECY-1930|| Light Strawberry, honey, ripe fruit with a tart, citrusy acidity after 6mo of aging. Creates an abundance of acidity quickly and haymay initially display a mousy-like tainted flavor but dissipates over time leading to ester production with hints of strawberry. May no longer be available Best used with ''Saccharomyces''; aging up to 6 months is recommended <ref name="ecy_website" />. ||Bantu beer brewery, South AfricaIsolated from Dr. Pepper

| NaardenensisBruxellensis ||''Brettanomyces naardenensisDekkera bruxellensis''||''B. naardenensisBrettanomyces bruxellensis''||ECY-30ECY35 || Strawberry, honey, Funky with barnyard notes accompanied by ripe tropical fruit with a tart, citrusy acidity after 6mo of aging. Creates an abundance of acidity quickly and may initially display a mousy-like tainted flavor but dissipates over time leading to ester production with hints of strawberry. Best used with ''Saccharomyces''; aging up to 6 months is recommended <ref name="ecy_website" />. ||Isolated from Drbottle of Belgian gueuze. Pepper

| Bruxellensis Anomala || ''Dekkera bruxellensisanomala'' || ''B. bruxellensisBrettanomyces anomalus'' || ECY35 ECY36 || Funky Notes of pineapple esters with acidity over time, no funk or barnyard notes accompanied by ripe tropical fruit present. Versatile <ref name="ecy_website" />. || Isolated from bottle of Belgian gueuze.

| Anomala || ''Dekkera anomala'' || ''B. anomalus'' || ECY36 || Notes of pineapple esters with acidity over time, no funk or barnyard present. Versatile <ref name="ecy_website" />. || |-| Lambicus || ''Dekkera bruxellensis || ''B. Brettanomyces bruxellensis'' || ECY37 || Cherry-stone and strong barnyard <ref name="ecy_website" />. ||

| Bruxellensis || ''Dekkera bruxellensis'' || ''B. 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>.|-| Claussenii || ''Dekkera anomala'' || ''Brettanomyces anomalus'' || Brett C || A strain of Brettanomyces clausenii, now understood to be among the Brettanomyces anomalus species. Minimal funk, tends to exhibit fruity pineapple or mango notes. Pairs well with fruit and/or hops. Recommended for secondary or co-fermentation as attenuation is variable. Attenuation: Highly Variable // Optimum Temp: 18-25ºC (64.4-77F) // Flocculation: Medium-low. Potentially no longer available (no longer listed on website) <ref name="escarpment_strains" />. ||

| Claussenii Bruxellensis || ''Dekkera anomalabruxellensis'' || ''B. anomalusBrettanomyces bruxellensis'' || Brett C D || A This strain of Brettanomyces clauseniibruxellensis is a notoriously vigorous fermentor, now understood to be among the Brettanomyces anomalus species. Minimal funk, tends to exhibit fruity pineapple suitable for primary fermentation of 100% Brett beers or mango notes. Pairs well with fruit and/or hops. Recommended for secondary or co-fermentation as attenuation where some extra funk is variabledesired. Attenuation: Highly Variable 80+% // Optimum Temp: 1820-25ºC (64.468-77F77ºF) // Alcohol tolerance: 12%+ // Flocculation: Medium-low. Potentially no longer available (no longer listed on website) <ref name="escarpment_strains" />. See [https://www.facebook.com/groups/MilkTheFunk/permalink/2331941676834033/?comment_id=2331958926832308&comment_tracking=%7B%22tn%22%3A%22R1%22%7D this MTF thread] on experiences using it for 100% fermentation. ||

| Bruxellensis Unknown || ''Dekkera bruxellensis'' Unknown || ''B. bruxellensis'' Unknown || Brett D M || This strain of Brettanomyces bruxellensis is a notoriously vigorous fermentor, suitable offers balanced funk fast. Suited for primary fermentation of 100% Brett use in sour beers or secondary fermentation where some extra funk is desired. Attenuation: 80+% // Optimum Temp: 20-25ºC (68-77ºF) // Alcohol tolerance: 12%+ // Flocculation: Medium, saisons, and other barrel-low aged treats <ref name="escarpment_strains" />. ||Michigan

| Bruxellensis || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || Brett Q || This strain typically completes primary fermentation within one month, and is also suitable for secondary aging of a wide range of beer styles where subtle Brett character is desired. Tasting notes include ripe strawberry, pear, apple, with underlying funk. Attenuation: 80%+ // Optimum Temp: 20-25ºC // Alcohol tolerance: High // Flocculation: Medium-low || Originally isolated from a barrel-aged sour beer from Quebec <ref name="escarpment_strains" />.

| Bruxellensis || ''Dekkera bruxellensis'' || ''B. Brettanomyces bruxellensis'' || Brussels Brett || This Brettanomyces bruxellensis strain displays a balanced selection of fruity characteristics, with testers noting plum, red berry, citrus, and red apple, alongside subtle acidity. It is suitable for primary or secondary fermentation, but does shine in secondary with extended aging, where it displays prominent funky flavours. Attenuation: 80+% // Optimum Temp: 22-25ºC // Alcohol tolerance: 12%+ // Flocculation: Medium-low <ref name="escarpment_strains" />. || |-| | Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || Berliner Brett II || Isolated from a bottle of Schultheiss Berliner Weisse, this ''B. bruxellensis'' strain produces notes of orchard fruit and pineapple. Complements Berliner Brett I nicely to produce a complete classic Berliner profile <ref name="escarpment_strains" />. ||

===[[Fermmento Labs]] (Brazil)Fermentis===

| Bruxellensis || ''Dekkera bruxellensis'' || ''BBrettanomyces bruxellensis'' || SafBrew™ BR-8 || || First known dried ''Brettanomyces'' product. Recommended for secondary fermentation; does not ferment dextrins. See [https://fermentis.com/en/product/safbrew-br-8/ the product page]. See [https://www.facebook.com/groups/MilkTheFunk/posts/7083324628362357/ this MTF post] on experiences using it for 100% fermentation (not recommended by vendor).|-|} ===[[Fermmento Labs]] (Brazil - CLOSED)==={| class="wikitable sortable"|-! Common Name !! Species Name !! Synonym Name !! Lab/Package !! Flavor/Aroma !! Source Note|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || FB2 Heavy Funkies || ||

===[[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>. ||

| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || TYB207 || This isolate exhibits good attenuation, and produces a moderate acidic-like character and an ester profile the combination of which produces a character reminiscent of sweet tarts. It's a fruity, funky tartness that's refreshing and crisp. This strain is well-suited for primary and secondary fermentation. Approximately 15 billion cells/vial. Temperature: 70 - 82 ºF 82ºF , Attenuation: 80%-82%, Flocculation: Medium-Low. || Isolated from a Belgian-inspired brewery in the Northeastern United States <ref>[http://www.theyeastbay.com/wild-yeast-and-bacteria-products/brettanomyces-single-strain-series-isolate-tyb-207 BRETTANOMYCES SINGLE STRAIN SERIES - ISOLATE TYB 207. The Yeast Bay website. Retrieved 11/16/2016.]</ref>.

| [[Propagate Lab]] || MIP-765 Native Brett Blend || Blend of ''Brettanomyces'' isolated from a number of natural sources. Isolates are from plums, natural wines, wild plants, and spontaneous fermentation. This blend will add complexity to any beer <ref name="propagate_website" />.|-| [[The Yeast Bay]] || Beersel || Not overly funky but there is a sweaty note hanging behind lemon and citrus fruits, nice blend of subtle funk and citrus/fruit. All strains were identified as ''B. bruxellensis'' <ref name="TYB_Blends">[https://www.facebook.com/groups/MilkTheFunk/permalink/1178127178882161/?comment_id=1178188512209361&offset=0&total_comments=9&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Nick Impellitteri regarding taxonomy of the TYB Brett blends. 11/13/2015.]</ref>. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer <ref name="TYB_starter_is_ok">[https://www.facebook.com/groups/MilkTheFunk/permalink/1551348668226675/?comment_id=1551707504857458&reply_comment_id=1551745278187014&comment_tracking=%7B%22tn%22%3A%22R922R%2313%22%7D Conversation on MTF with Nick Nick Impellitteri from The Yeast Bay. 01/16/2017.]</ref>.

| [[The Yeast Bay]] || Amalgamation II Brett Super Blend || Amalgamation II is a blend of 5 Brettanomyces isolates: Brettanomyces bruxellensis - Strain TYB184, Brettanomyces bruxellensis - Strain TYB207, Brettanomyces bruxellensis - Strain TYB261, and both Beersel Brettanomyces Blend isolates. The balanced funk of the Beersel isolates and TYB184, the sweet tart character of TYB207, and the tropical bouquet of the combined ester profile of lemon/pineapple/guava/mango/papaya contributed by all the isolates. Expect beers fermented with this blend to finish crisp, dry, tart and fruity with just a touch of funk on the finish. This blend produces noticeable character only 3-4 weeks into fermentation and is well suited for faster turnaround beers. Amalgamation II shines as a primary or secondary fermenter. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer <ref name="TYB_starter_is_ok" />. Reportedly a fast fermenter <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2071518542876349/ Łukasz Kierski. Milk The Funk Facebook group thread on TYB Amalgamation II. 04/25/2018.]</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 may be 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, it is advised 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.

Maintaining a temperature of 77°-86°F/25°-30°C results in faster growth than lower temperatures and is recommended <ref name="Trent_Preiss_Brett_Starters">[https://www.facebook.com/groups/MilkTheFunk/permalink/1334091429952401/?comment_id=1334157506612460&reply_comment_id=1334686406559570&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Mark Trent and Richard Preiss on optimal temperatures for Brett starters on MTF. 06/21/2016.]</ref>. ''Brettanomyces'' cell growth typically takes about 7-8 days to reach it's maximum growth <ref name="Yakobson_Propagation"></ref>, however some strains may grow at faster rates and finish in 3-4 days <ref>[[Brettanomyces_Propagation_Experiment]]</ref><ref name="Smith_2018" />. When the starter turns a rich creamy color, it should be done within a few hours after this visual indication occurs <ref name="Trent_Preiss_Brett_Starters"></ref>. Each step of a starter for Brett should be 7-8 days (or 3-4 days for faster growing strains).

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====

[[File:Martyniak.JPG|thumb|300px|right|Bright-field and fluorescent images of ''B. clausenii'', ''B. bruxellensis'', and ''B. lambicus''. The fluorescent images show the counting of the yeasts with and without declustering the bud and pseudohyphae. Source: https://link.springer.com/article/10.1007/s10295-016-1861-4. Used with permission from Brian Martyniak.]]The use of methylene blue, although popular in breweries, has been shown to be inaccurate when counting cells of ''Brettanomyces''. Trypan blue staining has been shown to give more accurate cell counting results than methylene blue <ref name="Hubbe" /><ref>[https://www.facebook.com/groups/MilkTheFunk/1407620505932826permalink/1451631908198352/?comment_id=14077236192558481451787331516143&comment_tracking=%7B%22tn%22%3A%22R822R5%22%7D Discussion with Richard Preiss regarding counting Brettanomyces with methylene blue versus trypan blue staining. 09/15/2016.]</ref>.

One of the problems with cell counting for ''Brettanomyces'' is that they tend to form pseudohyphae (elongated cells that are attached to one another) during growth. This makes counting the cells difficult. Martyniak et al. (2017) has proposed a new way of counting cells for ''Brettanomyces'' by using the fluorescence capability of a [https://www.nexcelom.com/nexcelom-products/cellometer-fluorescent-viability-cell-counters/cellometer-x2-fluorescent-automated-viability-cell-counter/ Nexcelom Cellometer where the X2 image cytometer] with stains created from acridine orange (AO) and propidium iodide (PI). The AO/-PI stains only the nuclei of the individual cells , allowing them to be counted easily by the cytometer regardless of pseudohyphae formation. During their research, they found that a "clausenii" strain of ''B. anomalus'' formed more pseudohyphae than two strains of ''B. bruxellensis'' (one of which was refered to as a "lambicus" strain), and this corresponded with higher viability over time. It was therefore hyopthesized that pseudohyphae might play a role in the longer survival of some strains of ''Brettanomyces'' over others <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1362220137139530/ Brian Martyniak. Milk The Funk thread on counting ''Brettanomyces'' cells. 06/29/2016.]</ref><ref>[https://link.springer.com/article/10.1007/s10295-016-1861-4 A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry. Brian Martyniak, Jason Bolton, Dmitry Kuksin, Suzanne M. Shahin, Leo Li-Ying Chan. 2017.]</ref><ref>[https://www.mbaa.com/meetings/archive/2017/proceedings/Pages/91.aspx 91. A novel concentration and viability detection method for Brettanomyces using image cytometry. Leo Chan. MBAA Poster. Retrieved 07/19/2018.]</ref>. It has been observed that pseudohyphae might be less common in ''Brettanomyces'' cultures that have been grown in wort versus lab media, making them easier to count <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1362220137139530/?comment_id=1932020246826180&comment_tracking=%7B%22tn%22%3A%22R%22%7D Caroline Whalen Taggart. Milk The Funk thread on Brettanomyces cell counting. 12/26/2017.]</ref>, but this method makes it easy to count cells which are attached via pseudohyphae. This method has been criticized for its relatively high cost (both for the fluorescent microscope and the dyes) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2201177583243777/ Justin Amaral, Jimmy Healy, Shawn Savuto, Matt Jumbard. MTF Facebook thread on the cost of using fluorescent micscopes and dyes for easier counting of ''Brettanomyces'' cells. 07/27/2018.]</ref>. See also:* [http://masterbrewerspodcast.com/092-automated-brettanomyces-cell-counts MBAA Podcast interview with Leo Chan on automating cell counting with of ''Brettanomyces'' psuedohyphae.]

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]]