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[[Image:Brettanomyces.jpg|thumb|200px|right|Brettanomyces]][[File:Brett-aroma-wheel.jpeg|thumb|200px|right|Brett Aroma Wheel by Dr. Linda Bisson and Lucy Joseph at UC Davis]]
'''''Brettanomyces''''', also referred to by brewers as "Brett" or "Bretta", is Greek for "British Fungus" and is a yeast that was originally thought of as an important yeast for producing the character of some 17th century and prior English ales. Since the wide adoption of pure cultures of ''Saccharomyces cerevisiae'' and ''S. pastorianus'' in the brewing and wine industries starting in the late 1800's, ''Brettanomyces'' has been mostly viewed as a spoilage yeast, except in Belgian lambic, Flanders red/brown beers, and a handful of styles of wine. More recently ''Brettanomyces'' has gained popularity in the United States (and subsequently the brewing industries of other countries) as a yeast that can contribute desirable and novel characteristics to beer and other alcoholic beverages. The genus name ''Dekkera'' is used interchangeably with ''Brettanomyces'', as it describes the teleomorph or spore-forming form of the yeast, although this form is extremely rare or perhaps even non-existent <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. Both genera evolved independently to ferment sugar and produce ethanol , around the same time that the first mammals emerged <ref name="SchifferdeckerRozpędowska">[httphttps://onlinelibrarywww.wileynature.com/doiarticles/10.1002/yea.3023/pdf The wine and beer yeast Dekkera bruxellensisncomms1305 Rozpędowska, E. Anna Judith Schifferdecker, Sofia DashkoHellborg, Olena PL. , Ishchuk, O. et al. Parallel evolution of the make–accumulate–consume strategy in Saccharomyces and Jure PiškurDekkera yeasts. 7 July 2014Nat Commun 2, 302 (2011).]<https://ref><doi.org/10.1038/ncomms1305.]</ref>. Both genera evolved independently to ferment sugar and produce ethanol <ref name="Gounot_2019Schifferdecker">[httpshttp://wwwonlinelibrary.biorxivwiley.orgcom/contentdoi/10.11011002/826990v1yea.full High complexity 3023/pdf The wine and degree of genetic variation in Brettanomyces beer yeast Dekkera bruxellensis population. Jean-Sébastien GounotAnna Judith Schifferdecker, Cécile NeuvégliseSofia Dashko, Kelle COlena P. FreelIshchuk, Hugo Devillers, Jure and Jure Piškur, Anne Friedrich, Joseph Schacherer. 20197 July 2014. DOI: https:]<//doi.ref><ref name="Gounot_2019">[https://www.biorxiv.org/content/10.1101/826990 826990v1.]</ref>. Although first isolated in 1889 full High complexity and again degree of genetic variation in 1899 by scientists at Guinness, the discovery of ''Brettanomyces'' was first publicly published by the Director of laboratory of the New Carlsberg Brewerybruxellensis population. Jean-Sébastien Gounot, Hjelte ClaussenCécile Neuvéglise, Kelle C. Freel, Hugo Devillers, Jure Piškur, in 1904 after he cultured it in 1903 from English beers that exhibited a sluggish secondary fermentation <ref>[httpAnne Friedrich, Joseph Schacherer. 2019. DOI: https://barclayperkinsdoi.blogspotorg/10.com1101/2013826990 .]</06/when-was-brettanomyces-discovererdref>.html "When was Brettanomyces discovered?" Ron Pattenson Although first isolated from beer in 1889 by H. Shut Up About Barclay Perkins blogSeyffert of the Kalinkin Brewery in St. 06/29/2013Petersberg and again in 1899 by J. retrieved 08/18/2016W.]</ref><ref>[http://breweryhistory.com/journal/archive/149/Yeast.pdf Ray Anderson. "ONE YEAST OR TWO? PURE YEAST AND TOP FERMENTATION"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. The Brewery History Society. 2012.]</ref>. At At the time of discovery, Claussen was aiming to recreate he included these newly discovered yeasts in the flavor profile of traditional English ales by fermenting them with pure cultures of genus ''Saccharomyces'', and either pitching pure cultures of his newly discovered ''Brettanomyces'' yeast along with ''SaccharomycesTorula''<ref name="Gilliland_1961">[https://crescentcitybrewtalk.com/brettanomyces-i/ "BRETTANOMYCES I OCCURRENCE, CHARACTERISTICS, or as he preferredAND EFFECTS ON BEER FLAVOUR" by R. B. Gilliland, after the primary fermentation of ''Saccharomyces'' <ref>[https://wwwB.A., B.Sc, F.facebookR.com/download/448702618652516/GB190328184AI.pdf "Improvements in and connected with the Manufacture of English Beers or Malt Liquors and in the Production of Pure Yeast Cultures for use thereinC." Patent application by Hjelte Claussen for ''Brettanomyces''(Arthur Guinness Son & Co. (Dublin) Ltd. A, St.DJames’s Gate, Dublin). 1903Received 21st Janurary, 1961.]<See also [http://ref>barclayperkins. ''Brettanomyces'', along with [[Hops#The_Freshening_Power_of_the_Hop_blogspot.28Hop_Creepcom/2013/06/when-was-brettanomyces-discovererd.29|dry hop creep]], html "When was identified as the source of secondary fermentation during long aged ales, contributing to their lasting high carbonation <ref>[https:Brettanomyces discovered?" Ron Pattenson. Shut Up About Barclay Perkins blog. 06/29/archive2013.org Retrieved 08/details18/principlespracti00syke "The principles and practice of brewing" Sykes, Walter John2016.]</ref><ref>[http://breweryhistory. London, Ccom/journal/archive/149/Yeast. Griffin and Company, limited, 1907pdf Ray Anderson. Pgs 384-388 "ONE YEAST OR TWO? PURE YEAST AND TOP FERMENTATION". The Brewery History Society. 2012.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/4709953772366133 Gareth Young. Milk The Funk Facebook group thread about . At the time of discovery, Claussen was aiming to recreate the flavor profile of traditional English brewers historically relying on Brettanomyces ales by fermenting them with pure cultures of ''Saccharomyces'', and dry hop creep for carbonation in long aged ales. 06/17/2021.]</ref>. Although Claussen saw the character from either pitching pure cultures of his newly discovered ''Brettanomyces'' as a desirable character in ales and identified its character as a quality of traditional English ales, as pure cultures of yeast along with ''Saccharomyces'' were introduced in English brewing in , or as he preferred, after the early 20th century, primary fermentation of ''BrettanomycesSaccharomyces'' became identified as a contaminate <ref>[https://www.facebook.com/download/448702618652516/GB190328184A.pdf "Improvements in both wineries and breweries due to some connected with the Manufacture of English Beers or Malt Liquors and in the phenols, acids, and haze that it sometimes producesProduction of Pure Yeast Cultures for use therein. " These phenols and acids have generally been described as "barnyard"Patent application by Hjelte Claussen for ''Brettanomyces''. A.D. 1903.]</ref>. ''Brettanomyces'', "burnt plastic"along with [[Hops#The_Freshening_Power_of_the_Hop_.28Hop_Creep.29|dry hop creep]], "wet animal", "fecal"was identified as the source of secondary fermentation during long aged ales, 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_2016contributing to their lasting high carbonation <ref>[https://archive.org/details/principlespracti00syke "The principles and practice of brewing" Sykes, Walter John. London, C. Griffin and Company, limited, 1907. Pgs 384-388.]</ref><ref name="Schifferdecker" /><ref name="Lucy_2015">>[httphttps://www.ajevonlinefacebook.orgcom/contentgroups/66MilkTheFunk/3permalink/379 4709953772366133 Gareth Young. Milk The Funk Facebook group thread about English brewers historically relying on Brettanomyces bruxellensis Aroma-Active Compounds Determined by SPME GC-MS Olfactory Analysisand dry hop creep for carbonation in long aged ales. C06/17/2021.M. Lucy Joseph, Elizabeth A. Albino, Susan E. Ebeler, Linda F. Bisson. 2015.]]</ref></ref>[https://www. youtube.com/watch?v=9BwO7gbhdns Martyn Cornell interview on Craft Beer Channel, "The general viewpoint of brewers (other than Lambic and Flanders redTime Is Now – reinventing the English IPA". 09/28/2022.]</brown brewers ref>(8 minutes in Belgium) and vintners that ''Brettanomyces. Beer historian, [https://barclayperkins.blogspot.com/search?q=brettanomyces Ron Pattinson], has stated that ''Brettanomyces'' is primarily a spoilage organism held for many decadeswas typically present in 1800's English aged beers such as stock ales, pale ales, porters, and still holds in most cases. More recently the positive flavor components barrel aged IPA's that have been identified were shipped to India, and it was considered an important component of both the flavor profile of these beers and in protecting beer from contaminants via ''Brettanomyces'' beer such as pineapple, stone fruits, and to some degree acetic acid, have regained popularity fermenting the majority of residual sugars <ref>[https://www.crowdcast.io/e/IPA-Past-Present-Future/1 Ron Pattinson. "History of IPA -1700s to 2021". Doug Piper's interview with brewers outside of BelgiumRon Pattinson. Some winemakers and wine tasters have also described wines with certain flavor compounds derived from ''Brettanomyces'' as positive characteristics of some wines <07/25/2021.]</ref> (~56 and 59 mins in )<ref>[https://dailywww.sevenfiftyfacebook.com/the-everything-guide-to-brettanomycesmilkthefunkthepodcast/videos/ "The Everything Guide to Brettanomyces1097016944410369 Ron Pattinson." GuildSomm. April 15, 2018MTF Live. Retrieved 0302/0410/20212022.]</ref>(45 minutes in). It is important to keep Following the discovery of this yeast by Claussen, isolates of ''Brettanomyces'' were discovered in Belgian lambic beers in mind that individual tasters on tasting panels describe some flavor compounds the 1920's. At this time, ''Brettanomyces'' was proposed as "negative" while others describe the new genus name, separating them as from the genus ''Torula'' <ref name="positiveGilliland_1961" (and sometimes a mixed response is given by a taster in regards to a certain flavor compound)/>. The species name 'bruxellensis', and this discrepancy in interpretations of meaning 'Brussels'Brettanomycesin Latin, became the proposed species name for ''B. bruxellensis.'' derived flavor compounds appears to be based on personal preference and experience. In some cases, for example for vinyl phenolsThis yeast species was then isolated from other industrial fermentations such as wine, cider, kombucha, low levels that are not detectable by some peoplekefir, but detected by others contribute positively to wineolives, while higher amounts contribute negativelyand bioethanol production. Custers was the first to attempt to describe the rest of the genus using phenotypic characteristics in 1940. ThusIn 1960, a lower intensity J. van der Walt observed some species of some flavor compounds can be seen as more ''Brettanomyces'' formed ascospores, and this form of ''Brettanomyces'' was named ''Dekkera''. However, after the initial discovery of sporulating strains of ''Brettanomyces'', this behavior has not been reported since, therefore some scientists prefer to use the term "''Brettanomyces''" to refer to this genus. Originally, a total of 9 species were attributed to the genus ''Brettanomyces'', but after gene technology was invented, some of these species were changed (see [[Brettanomyces#Taxonomy|Taxonomy]] below) <ref name="Stenseels_2015_Essential">[https://www.academia.edu/19646963/Brettanomyces_Bruxellensis_Essential_Contributor_in_Spontaneous_Beer_Fermentations_Providing_Novel_Opportunities_for_the_Brewing_Industry Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry. Jan Steensels. BrewingScience, Sept/Oct 2015 (Vol. 68). 2015.]</ref>. Although Claussen and others saw the character from ''Brettanomyces'' as a desirablecharacter in English ales and identified its character as a hallmark quality of traditional English ales, as pure cultures of ''Saccharomyces'' were introduced in English brewing in the early 20th century, ''Brettanomyces'' became identified as a contaminate in both wineries and breweries due to some of the phenols, acids, and haze that it sometimes produces. These phenols and acids have generally been described as "barnyard", "burnt plastic", "wet animal", "fecal", and "horse sweat", although some tasters describe these flavors with different terminology because they perceive certain flavor compounds differently while some other tasters simply cannot detect certain flavor compounds at all <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>. 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'' is primarily a spoilage organism, and this still holds in most cases today. More recently, however, the positive flavor components that have been identified in ''Brettanomyces'' beer such as "pineapple", "stone fruits", and to some degree acetic acid, have regained popularity with brewers outside of Belgium. Some winemakers and wine tasters have also described wines with certain flavor compounds derived from ''Brettanomyces'' as positive characteristics of some wines <ref>[https://daily.sevenfifty.com/the-everything-guide-to-brettanomyces/ "The Everything Guide to Brettanomyces." GuildSomm. April 15, 2018. Retrieved 03/04/2021.]</ref><ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/nzg.12368 Siimes, N. (2023). Having a drink with awkward Brett: Brettanomyces, taste(s) and wine/markets. New Zealand Geographer, 1– 11. https://doi.org/10.1111/nzg.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 completely largely subjective <ref name="Lucy_2015" /><ref>[https://www.guildsomm.com/public_content/features/articles/b/kelli-white/posts/brettanomyces Brettanomyces: Science & Context. Kelli White. GuildSomm website. 03/29/2018. Retrieved 05/10/2019.]</ref>.
See also:
* [https://www.facebook.com/groups/MilkTheFunk/permalink/3364694553558735/ A collection of early 1900's papers by Claussen and Alfred Chapman; MTF post by Cory Widmayer.]
* [https://beerandbrewing-com.cdn.ampproject.org/c/s/beerandbrewing.com/amp/brasserie-de-la-sennes-yvan-de-baets-explains-saisons-greatest-myth-the Yvan de Baets believes that ''Brettanomyces'' probably played an important role in historical saison.]
* [https://www.facebook.com/oddbreedwildales/videos/483806376609910 Presentation by Ron Pattinson on the role of ''Brettanomyces'' in historical English beer; hosted by Odd Breed Wild Ales on 02/15/2022.]
===Taxonomy===
It is common in scientific literature to see the names ''Dekkera'' and ''Brettanomyces'' used as the genus name, with ''Dekkera'' being the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph teleomorph] version and ''Brettanomyces'' being the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph anamorph]. There are five species within the genus of Brettanomyces: ''B. anomalus'', ''B. bruxellensis'', ''B. custersianus'', ''B. nanus'', and ''B. naardenensis'' (one study on the genetics of ''B. nanus'' from 1990 classified ''B. nanus'' as belonging to another genus of yeast called ''Eeniella'', however this has not been agreed upon in more recent studies <ref>[http://onlinelibrary.wiley.com/doi/10.1002/yea.320060403/full Dekkera, Brettanomyces and Eeniella: Electrophoretic comparison of enzymes and DNA–DNA homology. Maudy Th. Smith, M. Yamazaki, G. A. Poot. 1990.]</ref><ref>[https://theyeasts.org/details/72/2050 The Yeasts website. "Brettanomyces nanus". Retrieved 11/05/2022.]</ref><ref>[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=13366&lvl=3&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep=1&srchmode=1&unlock NCBI Taxonomy Browser. "Brettanomyces". Retrieved 11/05/2022.]</ref>). The species previously known as ''B. intermedius'' and ''B. lambicus'' have recently been genetically analyzed and reclassified as strains of ''B. bruxellensis'' <ref name="Agnolucci_2017">[https://link.springer.com/article/10.1007/s11274-017-2345-z Brettanomyces bruxellensis yeasts: impact on wine and winemaking. Monica Agnolucci, Antonio Tirelli, Luca Cocolin, Annita Toffanin. 2017.]</ref>. Of these five species, only ''B. anomalus'' and ''B. bruxellensis'' have been identified to have a teleomorph version. In their teleomorph version they are referred to as ''Dekkera anomala'' and ''Dekkera bruxellensis'' <ref name="smith_divol_2016"></ref><ref name="Schifferdecker"></ref><ref name="Steensels"></ref><ref name="Crauwels_2014">[http://aem.asm.org/content/80/14/4398.full Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Sam Crauwels, Bo Zhu, Jan Steensels, Pieter Busschaert, Gorik De Samblanx, Kathleen Marchald, Kris A. Willems, Kevin J. Verstrepen and Bart Lieven. 2014.]</ref>. All of the other names such as the ones often used by yeast labs (e.g. "claussenii") are derived from old nomenclature that is no longer used scientifically ([http://www.sciencedirect.com/science/article/pii/S0168160515001865#t0005 click here] for a table that lists old and new taxonomical nomenclature). Most ''Brettanomyces'' cultures from brewer's yeast labs are classified genetically as ''B. bruxellensis'' or ''B. anomalus''.
Recently a new species of ''Brettanomyces'' has been proposed, although classification has not been fully established. The proposed name is ''Brettanomyces acidodurans'' sp. nov. Two strains of ''B. acidodurans'' were isolated from olive oil from Spain and Israel; however, its presence in olive oil has been described as "rare" because only two strains were found after searching dozens of olive oils. Its closest relation is to ''B. naardenesis'' by 73% of its genetic makeup. No teleomorph form was observed. This species is a strong acetic acid producer, and it is very tolerant of acetic acid in its environment. It can consume lactose and cellobiose but does not consume maltose. it is unknown but a possibility that this species contributes to the vinegary taste of spoiled olive oils, although this has generally been attributed to acetic acid bacteria <ref>[https://www.ncbi.nlm.nih.gov/pubmed/28160110 Brettanomyces acidodurans sp. nov., a new acetic acid producing yeast species from olive oil. Péter G, Dlauchy D, Tóbiás A, Fülöp L, Podgoršek M, Čadež N. 2017.]</ref>.
* [http://suigenerisbrewing.com/index.php/2014/12/15/brett-trois-a-riddle-wrapped-in-a-mystery-inside-an-enigma/ "Brett Trois – A riddle, wrapped in a mystery, inside an enigma," Sui Generis Blog; an example of ''S. cerevisiae'' appearing like ''Brettanomyces'' cells under a microscope.]
* [https://bootlegbiology.com/diy/microbe-portrait-gallery Morphology examples on Bootleg Biology's website.]
===Culturing===
See [[Laboratory_Techniques#Brettanomyces|Laboratory Techniques]].
===Environment and Survival===
''Brettanomyces'' is commonly isolated from has been thought to occur naturally on the surface skins of wood structures within breweries, wineries, fruit such as apples and sometimes cideriesgrapes. These include structures such as wooden fermentation vesselsHowever, walls there are only a handful of the building, as well as the inside surface reports of wood barrels and actually buried within the wood of barrels. ''Brettanomyces'' has been easily cultured from within being identified on the wood skins of oak barrels up to 4 mm into the wood, and occasionally as deep as 5 to 8 mmfruit, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and one study found that the some cases where ''Brettanomyces'' was able to penetrate the French oak barrels up to 8 mmhas been found, its abundance is extremely minimal <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.154 Lentz, M., Putzke, T., Hessler, R. and Luman, while only penetrate American oak barrels up to 4 mmE. (2014) , Genetic and physiological characterization of the barrel yeast isolated from ripe fruit and analysis of fermentation and brewing potential, J. Inst. Brew., 120: 559– 564. DOI: 10.1002/jib.154.]</ref name="Agnolucci_2017" /><ref name="Cartwright_2018Comitini">[httphttps://www.ajevonlinefrontiersin.org/contentarticles/early10.3389/2018/05/23/ajevfmicb.20182019.18024 Reduction 00415/abstract Occurrence of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steamon grape berries and in related winemaking cellar. Zachary M. CartwrightFrancesca Comitini, Lucia Oro, Laura Canonico, Dean A. GlaweValentina Marinelli, Charles G. EdwardsMaurizio Ciani. 20182019. DOI: 10.53443389/ajevfmicb.20182019.1802400415.]</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 SO<subref name="Renouf_2007">2<[https://sub> concentrations)www.sciencedirect. Some strains are able com/science/article/pii/S0944501306000231?via%3Dihub Development of an enrichment medium to utilize the cellulose of the wood as detect Dekkera/Brettanomyces bruxellensis, a carbon sourcespoilage wine yeast, and occasionally form pseudohyphae within the wood which expands on the surface area of the cells allowing them more access to nutrients and allowing them to survive in nutrient deficient environments grape berries. Vincent Renouf, Aline Lonvaud-Funel. 2007. DOI: https://doi.org/10.1016/j.micres.2006.02.006.]</ref name="Cartwright_2018" />. Ozone gas has been shown to be an effective way to kill 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 primary source for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of ''Brettanomyces'' on wine grapes. Liquid ozone has been shown to be less effective at eliminating In addition, ''Brettanomyces''. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing ''Brettanomyces'' within isolated in abundance from the wood surfaces of barrels (see equipment/processed materials in wineries and breweries <ref name="smith_divol_2016" /><ref name="Schifferdecker" /><ref name="Loureiro_2003">[[Barrel#Sanitizing|Barrel Sanitationhttps://www.ncbi.nlm.nih.gov/pubmed/12892920 Spoilage yeasts in the wine industry. Loureiro V, Malfeito-Ferreira M. 2003.]] for information on pasteurizing barrels) </ref><ref name="Steensels" /><refname="Barata_2008">[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation 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). For example, an ongoing survey of wild yeasts in different regions of United States wilderness areas which isolated nearly 2,000 isolates with 262 unique species has not yet found a single occurrence of ''Brettanomyces'' in the wild (so far they have only surveyed non-human inhabited wild areas of the US and Alaska; substrates sampled included leaves, soil, bark, moss, mushrooms, needles, pine cones, twigs/wood, and other plant matter) <ref>[https://www.sciencedirectbiorxiv.comorg/sciencecontent/article10.1101/pii/S1466856417310068 Control of Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water2021.07.13.452236v1 Substrate, temperature, ozonated water and gaseous ozonegeographical patterns among nearly 2,000 natural yeast isolates. William J. Spurley, Kaitlin J. Fisher, Quinn K. Francesco CraveroaLangdon, Kelly V. Buh, Martin Jarzyna, Max A. B. Haase, Kayla Sylvester, Vasileios EnglezosRyan V. Moriarty, Kalliopi RantsiouDaniel Rodriguez, Fabrizio TorchioAngela Sheddan, Simone GiacosaSarah Wright, Susana Río SegadeLisa Sorlie, Vincenzo GerbiAmanda Beth Hulfachor, Luca RolleDana A. Opulente, Luca CocolinChris Todd Hittinger. 2018bioRxiv 2021.07.13. DOI452236; doi: https://doi.org/10.10161101/j.ifset2021.201807.0313.017452236.]</ref>. Although the role of It is therefore unclear that ''Brettanomyces'' appears to be limited in distillation, found on grape skins originated there or from the industrial processing where it has been isolated during the fermentation process of tequila makingis more abundant. It has 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 already contaminated with ''Brettanomyces'' <ref>[https://youtu.be/G2nhUM5PIrg?t=309 Dr. Bryan Heit. BotB - Where (Do) The Wild Brettanomyces Roam?. ~5 mins in. Retrieved 07/10/2022.]</ref><ref name="Renouf_2007" /><ref name="Steensels">[http://www.sciencedirect.com/science/article/pii/S0168160515001865 Brettanomyces yeasts — From spoilage organisms to valuable contributors to sanitizeindustrial fermentations. The survivability Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.]</ref><ref name="Barata_2008" /><ref name="Loureiro_2003" />. ''Brettanomyces'' has also partly been attributed is known to be difficult to its ability grow in a lab due to form slow growth, specific nutrient requirements, or perhaps because of a "VBNC" state (see [[Quality_AssuranceWild_Yeast_Isolation#BiofilmsWild_Brettanomyces|biofilmWild ''Brettanomyces'']] (in particular for more information), which may account for the lack of evidence for fruit being the primary natural habitat for ''B. bruxellensisBrettanomyces''). Microorganisms that can form a biofilm are More recently, techniques have been invented to more resistant to chemical cleaning agents easily isolate and sanitizers than those that dongrow ''Brettanomyces''t<ref name="Renouf_2007" /><ref name="Comitini" />. There is also significant evidence that the natural habitat of ''Brettanomyces'' has therefore been identified might actually be the root systems of certain plants, known as a significant contaminate for breweries the [https://www.nature.com/scitable/knowledge/library/the-rhizosphere-roots-soil-and wineries-67500617/ "rhizosphere"]. Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''BThe rhizosphere refers to the complex symbiotic community of microbe populations that live on and around the root system of plants. bruxellensis''. 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 rhizosphere, essential oil and mineral nutrients uptake in olive productiondill/common bean intercropping system. Ann Appl Biol, 169: 384-397. https://doi.org/10.1111/aab.12309.]</ref><ref>[https://archive.aessweb.com/index.php/5003/article/view/3333 I.O, lemonadeS. ., kombucha& G.P, yogurtO. . (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.S. Olahan, I.O. Sule, T Garuba, Y.A. Salawu. Science World Journal. 2016.]</ref><ref>[https://ojs.unud.ac.id/index.php/jbb/article/view/36023 NOERFITRYANI, Noerfitryani; HAMZAH, Hamzah. ''BTHE 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.v3i1. June 20165613.]</ref>. See Dr. Bryan Heit's video [https://www.youtube.com/watch?v=G2nhUM5PIrg "Where (Do) The Wild Brettanomyces Roam?"] and [https://www.facebook.com/groups/MilkTheFunk/posts/5940213029340195 his comments in Milk The Funk], as well as [https://www.youtube.com/watch?v=BrR7G_YyfmA "Philip Poole. Plant Control of the Rhizosphere Microbiome"]. For documented isolation attempts from plant rhizospheres, see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild Yeast Isolation]].
The occurrence of ''Brettanomyces'' has been more commonly identified in industrial food processing areas (wine, beer, kombucha, soft drinks, dairy products, tea, sourdough, etc.) <ref name="Crauwels_2016">[https://academic.oup.com/femsyr/article-abstract/17/1/fow105/2670560/Fermentation-assays-reveal-differences-in-sugar?redirectedFrom=fulltext Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains. Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains. Sam Crauwels, Filip Van Opstaele, Barbara Jaskula-Goiris, Jan Steensels, Christel Verreth, Lien Bosmans, Caroline Paulussen, Beatriz Herrera-Malaver, Ronnie de Jonge, Jessika De Clippeleer, Kathleen Marchal, Gorik De Samblanx, Kris A. Willems, Kevin J. Verstrepen, Guido Aerts, and Bart Lievens. 2016]</ref>. For example, ''B bruxelensis'', ''B. anomala'', and ''B. custersianus'' have mostly been isolated from wine or beer production, while ''B. naardenensis'' has mostly been isolated from soda production <ref name="Tiukova_2019">[https://www.mdpi.com/2076-2607/7/11/489 Assembly and Analysis of the Genome Sequence of the Yeast Brettanomyces naardenensis CBS 7540. Ievgeniia A. Tiukova, Huifeng Jiang, Jacques Dainat, Marc P. Hoeppner, Henrik Lantz, Jure Piskur, Mats Sandgren, Jens Nielsen, Zhenglong Gu, and Volkmar Passoth. 2019. DOI: https://doi.org/10.3390/microorganisms7110489.]</ref>. ''Brettanomyces'' is not considered to be airborne; however, one study has demonstrated a very small amount of cells in the air at wineries where wine with ''Brettanomyces'' in it was being handled (most of the yeasts found in the air were ''Aureobasidium'' and ''Cryptococcus'', which aren't considered spoilage organisms in beer and wine). This set of studies also determined that very specific methodology was needed in order capture ''Brettanomyces'' from the air, and indicated that the yeast was "stressed". While it is possible for ''Brettanomyces'' to be briefly carried by gusts of air, it only happens in the vicinity where the ''Brettanomyces'' beer or wine is being bottled (more so) or is actively fermenting (less so) <ref>[http://www.sciencedirect.com/science/article/pii/S0956713513002284 Screening of yeast mycoflora in winery air samples and their risk of wine contamination. E. Ocón, P. Garijo, S. Sanz, C. Olarte, R. López, P. Santamaría, A.R. Gutiérrez. Food Control Volume 34, Issue 2, December 2013, Pages 261–267.]</ref>. Good cleaning and sanitation and cold temperatures should be employed to keep ''Brettanomyces'' from contaminating other equipment; however, flying insects are also a potential cause for contamination of ''Brettanomyces'' (although evidence for this is lacking). ''Brettanomyces'' is commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries (although the median occurrence of ''Brettanomyces'' in barrels may be very low to none within a given winery or brewery depending on their hygiene and other factors <ref>[https://link.springer.com/article/10.1007/s00217-011-1523-8 Guzzon, R., Widmann, G., Malacarne, M. et al. Survey of the yeast population inside wine barrels and the effects of certain techniques in preventing microbiological spoilage. Eur Food Res Technol 233, 285–291 (2011). https://doi.org/10.1007/s00217-011-1523-8.]</ref><ref>[https://agris.fao.org/agris-search/search.do?recordID=IT2007601151 Fontanot, S.; Ninino, M.E.; Comi, G.; Elimination of Dekkera/Brettanomyces from barriques of the Italian CDO Isonzo area. Controlled Designation of Origin; Friuli-Venezia Giulia. 2006.]</ref>). These include structures such as wooden fermentation vessels, walls of the building, as well as the inside surface of wood barrels and actually buried within the wood of barrels. ''Brettanomyces'' has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and occasionally as deep as 5 to 8 mm, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and one study found that the ''Brettanomyces'' was able to penetrate the French oak barrels up to 8 mm, while only penetrate American oak barrels up to 4 mm) of the barrel <ref name="Agnolucci_2017" /><ref name="Cartwright_2018">[http://www.ajevonline.org/content/early/2018/05/23/ajev.2018.18024 Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam. Zachary M. Cartwright, Dean A. Glawe, Charles G. Edwards. 2018. DOI: 10.5344/ajev.2018.18024.]</ref>, with the highest concentration of surviving cells being at the top staves where oxygen is more accessible (although Cartwright et al. found the opposite was true, perhaps due to methodology of sampling or a difference in SO<sub>2</sub> concentrations). Some strains are able to utilize the cellulose of the wood as a carbon source, and occasionally form pseudohyphae within the wood which expands the surface area of the cells allowing them more access to nutrients and allowing them to survive in nutrient deficient environments <ref name="Cartwright_2018" />. Ozone gas has been shown to be an effective way to kill ''Brettanomyces'' that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of ''Brettanomyces'' on wine grapes. Liquid ozone has been shown to be less effective at eliminating ''Brettanomyces''. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing ''Brettanomyces'' within the wood of barrels (see [[Barrel#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment. Fabrizio, Vigentini, Parisi,Picozzi, Compagno, Foschino. 2015.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1466856417310068 Control of Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and gaseous ozone. Francesco Craveroa, Vasileios Englezos, Kalliopi Rantsiou, Fabrizio Torchio, Simone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. Although the role of ''Brettanomyces'' appears to be limited in distillation, it has been isolated during the fermentation process of tequila making. It has also been isolated from drains, pumps, transfer hoses, and other equipment that is difficult to sanitize. The survivability of ''Brettanomyces'' has also partly been attributed to its ability to form a [[Quality_Assurance#Biofilms|biofilm]] (in particular ''B. bruxellensis''). Microorganisms that can form a biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. ''Brettanomyces'' has therefore been identified as a significant contaminate for breweries and wineries. Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''B. bruxellensis''. ''Brettanomyces'' is also commonly found in sherry, and is found (although only rarely) in olive production, lemonade, kombucha, yogurt, pickles, and soft drinks. ''B. anomalus'' and ''B. bruxellensis'' are generally found much more commonly than the other three species of ''Brettanomyces'' <ref name="smith_divol_2016">[http://www.sciencedirect.com/science/article/pii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Brendan D. Smith, Benoit Divol. June 2016.]</ref>. Unlike most genera of yeast, ''Brettanomyces'' has the characteristics of being very tolerant to harsh conditions, including high amounts of alcohol (up to 14.5-15% ABV <ref name="Crauwels1" /><ref name="Agnolucci_2017" />), a pH as low as 2 <ref>[http://www.winesandvines.com/template.cfm?section=news&content=141954 Wines and Vines. New Research on Role of Yeast in Winemaking; report on a presentation by David Mills and Lucy Joseph from UC Davis. 11/14/2014. Retrieved 08/16/2015.]</ref>, and environments with low nitrogen <ref name="Schifferdecker"></ref> and low sugar sources <ref name="Smith_2018">[https://www.sciencedirect.com/science/article/pii/S0740002017308249 The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium. Brendan D.Smith and Benoit Divol. 2018. DOI: https://doi.org/10.1016/j.fm.2017.12.011.]</ref>. It has been reported that ''B. bruxellensis'' is more tolerant of high levels of bicarbonate than compared to ''S. cerevisiae'' (levels above 100 mg/l slow the fermentation of ''B. bruxellensis'', but do not completely inhibit it, with up to 400 mg/l being tested in one study) <ref name="Thompson-Witrick_2022">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2021.1940654 Katherine A. Thompson-Witrick & Eric R. Pitts (2022) Bicarbonate Inhibition and Its Impact on Brettanomyces bruxellensis Ability to Produce Flavor Compounds, Journal of the American Society of Brewing Chemists, 80:3, 270-278, DOI: 10.1080/03610470.2021.1940654.]</ref>. It has been reported that some strains require a very low concentration of fermentable sugars (less than 300 mg/L) and nitrogen (less than 6 mg/L), which is less than most wines contain <ref name="Smith_2017">[https://www.sciencedirect.com/science/article/pii/S0740002017308249 The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium. Brendan D. Smith, Benoit Divol. 2017.]</ref>. Some strains are able to utilize ethanol, glycerol, acetic acid, and malic acid when no other sugar sources are available <ref name="Smith_2018" />. This capability allows ''Brettanomyces'' to survive in alcoholic beverages such as beer, wine, and cider. In alcoholic beverages, ''B. bruxellensis'' tends to lag after the primary fermentation with ''Saccharomyces''. It is believed that during this lag phase, ''B. bruxellensis'' adapts to the harsh conditions of the beverage (low pH, high concentrations of ethanol, and limited sugar/nitrogen sources). After this lag phase, ''B. bruxellensis'' can grow and survive when no other yeasts can. ''Brettanomyces'' is also more resistant to pH and temperature changes, and tolerant of environments limited in oxygen (although ''Brettanomyces'' prefers the availability of at least a little bit of oxygen). Scientifically, which specific nitrogen and carbon sources ''B. bruxellensis'' uses in these stressful environments has not received much research <ref name="smith_divol_2016"></ref>. [https://www.winesandvines.com/news/article/200000/New-Tools-to-Limit-Wine-Spoilage One study from Dr. Charles Edwards] found that a combination of keeping wine under 54°F (12.2°C) and alcohol at or above 14% resulted in a decline of ''B. bruxellensis'' populations for up to 100 days for two strains that were tested. The study found that neither of the strains grew well at 14% and stopped growth completely at 16% ABV in wine, but one strain grew better than the other at 15%, demonstrating the genetic diversity of ''Brettanomyces''. The researchers concluded that a combination of high ethanol and cold temperatures as well as sulfur dioxide, chitosan, and filtration could be used to control ''Brettanomyces'' in winemaking. ''Brettanomyces'' has been found to be able to grow at temperatures as low as 50°F (10°C) and as high as 95°F (35°C); see [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|fermentation temperature]] for more information <ref>[http://www.ajevonline.org/content/early/2017/01/05/ajev.2017.16102 Interactions between Storage Temperature and Ethanol that Affect Growth of Brettanomyces bruxellensis in Merlot Wine. Taylor A. Oswald, Charles G. Edwards. 2017.]</ref>. ''Brettanomyces'' is also tolerant of IBU's, and there is some evidence that ''Brettanomyces'' is only inhibited by very high IBU's. One study reported that one strain of ''B. bruxellensis'' was inhibited by exposure to 250 mg/L of isomerized hop extract (roughly 250 IBU). Very little inhibition occurred at 150 IBU and about a third of the cells were inhibited at 200 IBU. The inhibited cells were recoverable in YPD media treated with catalase enzyme. In comparison, ''S. cerevisiae'' can be inhibited by 500 mg/L of iso-alpha acids <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2022.902110/full "Transcriptome Analysis of Viable but Non-Culturable Brettanomyces bruxellensis Induced by Hop Bitter Acids". He Yang, Zhao Junfeng, Yin Hua, Deng Yuan. Frontiers in Microbiology. 2022. DOI: 10.3389/fmicb.2022.902110 .] See also [https://www.facebook.com/groups/MilkTheFunk/posts/7473091549385661/ this MTF post]</ref>.
The genetic diversity of ''Brettanomyces'' is particularly wide. For example, one study that analyzed the whole genomes of 53 strains of ''B. bruxellensis'' found that the overall genetic diversity between different strains of ''B. bruxellensis'' was higher than strains of ''S. cerevisiae'' (however, the entire gene set, known as the ''pangenome'', of all the genes among all of the strains of ''B. bruxellensis'' is much smaller than the entire gene set of ''S. cerevisiae'') <ref name="Gounot_2019" />. Some studies have indicated that strains of ''B. bruxellensis'' have adapted to specific environments. For example, one study found that strains of ''B. bruxellensis'' isolated from wine had 20 genes involved in the metabolism of carbon and nitrogen, whereas strains isolated from beer did not. This indicated that ''B. bruxellensis'' strains living in wine have adapted to the harsher environment of wine <ref name="smith_divol_2016"></ref>. Another study found that one out of the two strains tested that were isolated from soda could not ferment maltose, and only strains isolated from wine were able to grow in wine and the beer/soda strains did not. The wine strains were also more resistant to sulfites, which are commonly used in the wine industry to prevent microbial contamination <ref name="Crauwels_2016" />. The whole genome sequencing of one strain of ''B. naardenensis'' and lambic strains of ''B. bruxellensis'' found that they are missing the genes associated with nitrate utilization, indicating that the assimilation of nitrates is not required to survive in beer, perhaps because of the abundance of nitrogen from other sources found in beer <ref name="Tiukova_2019" /><ref name="colomer_2020_genome" />.
* [[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>.
==''Brettanomyces'' Metabolism==
Beta-glucosidases can break down the beta-glycosidic bond in disaccharides (cellulose, cellobiose, and gentiobiose) <ref name="ucdavis_chemwiki">[http://chemwiki.ucdavis.edu/Core/Organic_Chemistry/Carbohydrates/Disaccharides "Disaccharides." UC Davis Chemwiki. Retrieved 05/15/2016.]</ref><ref name="smith_divol_2016"></ref>, as well as glycosides. Glycosides are sugar molecules connected to other organic compounds such as acids, alcohols, and aldehydes which are flavor and aroma inactive due to the sugar molecule attached. By cleaving off the sugar molecule through beta-glucosidase activity, ''Brettanomyces'' species can liberate these compounds (called aglycones) into their aroma-active and flavor-active states, or states that may become flavor and aroma active through further modification <ref>Daenen et al., 2008. Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. FEMS Yeast Res. 8, 1103-1114.</ref>. Therefore some ''Brettanomyces'' strains are believed to be able to produce novel flavors and aromas from hops, fruits, and fruit pits that ''Saccharomyces'' yeasts cannot produce. In addition, the liberated aroma and flavor active compounds may be further processed by ''Brettanomyces'' through ester production or destruction pathways. See [[Brettanomyces#Glycosides_and_Beta-Glucosidase_Activity|Beta-Glucosidase Activity]] for more information.
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 (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 were generally better at fermenting cellobiose than strains isolated from beer at 15°C (59°F), however at 22.5°C (72.5°F) some of the beer strains started to utilize cellobiose, indicating that temperature plays a role in whether ''Brettanomyces'' can ferment certain sugars <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.]
Currently, research into how well ''Brettanomyces'' strains ferment the trisaccharide maltotriose has not been explored much by science. However, one study found that ''B. custersianus'' can ferment maltotriose. Another study found that all 7 strains of ''B. bruxellensis'' tested could ferment maltotriose, but not the trisaccharide raffinose. More investigation into this possibility is needed <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, one study by Tyrawa et al. found that several strains of ''B. bruxellensis'' fermented at 30°C "smelled terrible" of aromas typical of sulfur and autolysis <ref name=" Tyrawa_2017" /><ref>[https://www.facebook.com/groups/MilkTheFunk/posts/5684564058238428/?comment_id=5684866488208185&reply_comment_id=5692967664064734 Richard Preiss on very warm fermentation temperatures for ''Brettanomyces''. Milk The Funk Facebook group. 04/13/2022.]</ref>. At 20°C (68°F) fermentation rate is about half as slow. ''Brettanomyces'' will still grow at temperatures as low as 15°C (59°F) with about a third of strains being able to grow as low as 10°C (50°F) <ref name="Conterno_2006">[http://www.ajevonline.org/content/57/2/139 Genetic and Physiological Characterization of Brettanomyces bruxellensis Strains Isolated from Wines. Lorenza Conterno, C.M. Lucy Joseph, Torey J. Arvik, Thomas Henick-Kling, Linda F. Bisson. 2006.]</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/24290676 Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in Wine. Zuehlke JM, Edwards CG. 2013. DOI: 10.4315/0362-028X.JFP-13-243R.]</ref> but growth will be much slower. However, one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth and fermentation temperature range of 20-32°C. The growth rate at 15°C, while still slowly active, varies from strain to strain with some strains growing very poorly. Carbohydrates are consumed much slower, with cellobiose metabolizing ceasing for some strains (although phenol production stayed the same between 15°C and 22.5°C) <ref name="Tyrawa_2017" />. At a temperature of 35°C (95°F), fermentation is greatly inhibited due to cell death for most 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]].
The below table is an example of the variety of sugar types that different strains/species of ''Brettanomyces'' banked at the [https://catalogue.ncyc.co.uk National Collection of Yeast Cultures] can ferment under semi-aerobic fermentation and aerobic growth (the '''semi-aerobic''' fermentation value is probably more useful for brewers since oxygen availability is limited during fermentation in normal brewing practices):
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].
Esters are also broken down via a process called hydrolysis. Hydrolysis breaks the esters down using the same esterase enzyme within the ''Brettanomyces'' cells that are used to create esters. In general, all acetate based esters, except for phenethyl acetate and methyl acetate, are broken down faster than non-acetate esters by ''Brettanomyces''. In lambic brewing, sometime after the primary fermentation finishes, ''Pediococcus'' begins to produce lactic acid. The formation of lactic acid by ''Pediococcus'' coincides with the appearance and growth of ''Brettanomyces'', which produces more acetic acid. After another 2-3 months, the ester content of the lambic beer changes and reaches an equilibrium. Ethyl acetate and ethyl lactate are greatly increased, while isoamyl acetate is greatly decreased, reaching an equilibrium of these esters. Given a static amount of acetic acid, ''Brettanomyces'' reaches an equilibrium of ethyl acetate within 24 hours, while ethyl lactate equilibrium takes longer and is much more complex. In lambic, the majority of ester production and breakdown occurs within 1-3 months after lactic acid production by ''Pediococcus'' begins, and at a pH of around 3.5 and a temperature of around 15°C or less <ref name="Spaepen"></ref>. Pitching rate of ''Brettanomyces'' has an effect on the breakdown of isoamyl acetate with higher pitching rates breaking down this ester at a faster rate <ref name="MTF_Brett_Secondary"></ref>. As far as we are aware, ethyl acetate is not metabolized further by ''Brettanomyces'', and the level of ethyl acetate will not be hydrolized over time (although levels can continue to increase over time with more oxygen oxposure, since oxygen exposure encourages acetic acid synthesis by ''Brettanomyces'' and acetic acid bacteria, and acetic acid and ethanol are then metabolized into ethyl acetate by ''Brettanomyces'').
====Phenol Production====
[https://www.britannica.com/science/phenol Phenols ] such as 4-vinylphenol (4VP; barnyard, medicinal) and 4-vinylguaiacol (4-VG; clove) can be produced in beer through the decarboxylation of hydroxycinnamic acids (HCAs) by yeast, and also in small amounts by long boils with a portion of the wort coming from wheat (3+ hours resulted in 0.3 ppm of 4VG). HCAs, such as ferulic acid and p-coumaric acid, are found present in the non-starch polysaccharide arabinoxylan in malt malted barley and wheat. They are released into wort during mashing at levels that are far below their flavor thresholds (greater than 500ppm flavor threshold) <ref name="kalb_2021">[https://pubs.acs.org/doi/full/10.1021/acs.jafc.1c03018 Investigations into the Ability to Reduce Cinnamic Acid as Undesired Precursor of Toxicologically Relevant Styrene in Wort by Different Barley to Wheat Ratios (Grain Bill) during Mashing. Valerian Kalb, Torsten Seewald, Thomas Hofmann, and Michael Granvogl. Journal of Agricultural and Food Chemistry 2021 69 (32), 9443-9450. DOI: 10.1021/acs.jafc.1c03018.]</ref><ref name="lentz_2018">[http://www.mdpi.com/2311-5637/4/1/20/html#B13-fermentation-04-00020 The Impact of Simple Phenolic Compounds on Beer Aroma and Flavor. Michael Lentz. 2018. doi: 10.3390/fermentation4010020.]</ref>. Some strains of ''Oenococcus oeni'' and ''Lactobacillus'', as well as some strains of yeast such as ''Pichia'' spp, have been found to produce HCA's via cinnamoyl esterase activity in wine, although when these strains have been used in wine to increase the HCA levels, the final phenol levels produced by ''Brettanomyces'' were the same as wine that did not have an increase in HCA levels (the precursors in wine that lead to HCA's are different than they are in beer) <ref>[http://www.ajevonline.org/content/early/2018/05/02/ajev.2018.17092 Influence of Oenococcus oeni and Brettanomyces bruxellensis on Wine Microbial Taxonomic and Functional Potential Profile. Marie Lisandra Zepeda-Mendoza, Nathalia Kruse Edwards, Mikkel Gulmann Madsen, Martin Abel-Kistrup, Lara Puetz, Thomas Sicheritz-Ponten, Jan H. Swiegers, Am J Enol Vitic. May 2018. DOI: 10.5344/ajev.2018.17092.]</ref>. The esters in grape must that contain HCA's (ethyl ferulate and ethyl coumarate) can also be formed by acidic hydrolysis which occurs at the low pH of wine, and HCA's can then be released from these esters. This formation of esters is a slow process in wine, with one study reporting ~0.03 ppm of ethyl ferulate and ~0.4 ppm of ethyl coumarate at the end of primary fermentation and ~0.09 ppm of ethyl ferulate and ~1.4 ppm of ethyl coumarate after 10 months of barrel aging <ref>[https://pubs.acs.org/doi/full/10.1021/jf204908s Hydroxycinnamic Acid Ethyl Esters as Precursors to Ethylphenols in Wine. Josh L. Hixson, Nicola R. Sleep, Dimitra L. Capone, Gordon M. Elsey, Christopher D. Curtin, Mark A. Sefton, and Dennis K. Taylor. 2012. DOI: 10.1021/jf204908s.]</ref>. We are not aware of any studies that have reported an increase in HCA's from acidic hydrolysis over time in beer; however, this is a standard laboratory technique for forcing the release of HCA's from barley (although this lab technique uses a lower pH then that of sour beer). In addition, it has been demonstrated that spent yeast (''S. cerevisiae'' collected after beer fermentation) contains a small fraction of phenols and polyphenols absorbed from wort during fermentation <ref name="Cortese_2020">[https://www.sciencedirect.com/science/article/pii/S0021967319310295 Quantification of phenolic compounds in different types of crafts beers, worts, starting and spent ingredients by liquid chromatography-tandem mass spectrometry. Manuela Cortese, Maria Rosa Gigliobianco, Dolores Vargas Peregrina, Gianni Sagratini, Roberta Censi, Piera Di Martino. Journal of Chromatography A; Volume 1612, 8 February 2020, 460622. DOI: https://doi.org/10.1016/j.chroma.2019.460622.]</ref>. It is therefore conceivable that HCA levels could increase in sour beer over time.
While both ''Saccharomyces'' (only by "phenolic off flavor positive/POF+" strains) and ''Brettanomyces'' strains have varying capabilities based on strain of converting hydroxycinnamic acids to their vinyl derivatives <ref>[https://link.springer.com/article/10.1007/s10482-016-0793-3 González, C., Godoy, L. & Ganga, M.A. Identification of a second PAD1 in Brettanomyces bruxellensis LAMAP2480. Antonie van Leeuwenhoek 110, 291–296 (2017). https://doi.org/10.1007/s10482-016-0793-3.]</ref><ref name="Lentz">[http://www.mdpi.com/2304-8158/4/4/581/htm Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast. Michael Lentz and Chad Harris. 2015.]</ref><ref>[https://www.biorxiv.org/content/10.1101/2024.04.16.586637v1 Characterization of Brettanomyces bruxellensis phenolic acid decarboxylase enzyme expressed in E. coli. Michael R. Lentz. bioRxiv 2024.04.16.586637; doi: https://doi.org/10.1101/2024.04.16.586637.]</ref>, ''Brettanomyces'' is also able to reduce these vinyl phenol derivatives to ethyl phenol derivatives. Phenolic acid decarboxylase (PAD) is the enzyme that converts the HCAs into vinyl phenols. Vinyl reductase (VA) is the enzyme that reduces vinyl phenols to ethyl phenols <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1632316743463200/ Analysis of phenolic acid decarboxylase enzyme from the wine spoilage yeast Brettanomyces bruxellensis (poster). Mike Lentz, Jamie Lynch, Pricilla Walters, Rachel Licea, Henok Daniel, Kimberly Pereira. 2017.]</ref>. Phenol production has been observed to occur shortly after inoculation of ''Brettanomyces'' and has been hypothesized to play a large role in replenishing NAD<sup>+</sup> to alleviate the initial lag growth phase in ''Brettanomyces'' <ref name="Tyrawa_Masters">[https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation. University of Guelph, Masters Thesis. Department of Molecular and Cellular Biology. 2020.]</ref>. While almost all strains of ''Brettanomyces'' produce ethyl phenols, one strain of ''Brettanomyces anomalus'' has been found that has lost the genetic capability to produce phenols <ref name="colomer_2020_genome" />.
These vinyl derivatives have similar tastes to the ethyl derivatives but have lower flavor thresholds. Levels of all compounds produced vary depending on species and strain of ''Brettanomyces''. Although the production of ethyl phenols has been identified to occur higher in substrates with more available sugars, and this has also correlated with higher growth <ref name="Barata_2008">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00415.x/full The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine. André Barata, Daniela Pagliara, Tiziana Piccininno, Francesco Tarantino, Wilma Ciardulli, Manuel Malfeito-Ferreira, Virgílio Loureiro. 2008. DOI: 10.1111/j.1567-1364.2008.00415.x]</ref>, some data supports that pitching rate does not have an effect on how much phenol content is produced by ''Brettanomyces''<ref name="MTF_Brett_Secondary">[http://www.milkthefunk.com/wiki/Brettanomyces_secondary_fermentation_experiment Brettanomyces secondary fermentation experiment. Milk The Funk Wiki. Lance Shaner and Richard Preiss. Retrieved 04/21/2016.]</ref>. Additionally, Curtin et al. (2013) showed that while both cell growth and attenuation was inhibited in anaerobic conditions in wine, phenol production was not (in fact, the phenol production was inhibited by aerobic conditions). They also showed that each of the three strains of ''B. bruxellensis'' tested all produced the same amount of phenols, while other flavor compounds such as esters were produced at different levels by each of the strains <ref>[https://www.ncbi.nlm.nih.gov/pubmed/24010603 Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma. Curtin CD, Langhans G, Henschke PA, Grbin PR. 2013]</ref>. [https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert's Bachelors thesis] also reported no correlation between fermentation rate and phenol production in several strains of ''B. bruxellensis'' <ref name=Humbert_2021">[https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert for the degree of Honors Baccalaureate of Science in Chemical Engineering presented on May 21, 2021. Title: Performance of Brettanomyces Yeast Strains in Primary and Secondary Beer Fermentations.]</ref>. Perhaps growth itself is not as much of a factor in producing phenols, but having sugars available for metabolism is. This contradicts the somewhat popular belief that under-pitching ''Brettanomyces'' produces more "funky" flavors. Additionally, perhaps some strains are perceived as "funkier" than others due to less ester production and more fatty acid production (isobutyric acid, for example), rather than more phenol production.
Another study by Tyrawa et al. found that fermentation temperature also did not have a significant effect on phenol production in 9 strains of ''B. bruxellensis''. Given the same wort composition, strains of ''B. bruxellensis'' produced similar levels of phenols at both 15°C and 22.5°C. The ester production was affected by this temperature difference in some strains but not others (see [[Brettanomyces#Ester_Production|Esters]] above). Assuming that phenols contribute the "funky" flavor characteristics, this suggests that perhaps a lower balance of esters to phenols produces a more "funky" tasting beer more so than a beer with more phenol content. If so, a lower fermentation temperature may be one way to emphasize phenols over fruity esters <ref name="Tyrawa_2017" />. Both Tyrawa and Humbert reported that there was no correlation between flavor profiles from phenol production of different strains of ''Brettanomyces bruxellensis'' and their origin <ref name=Humbert_2021" />.
* [http://phenol-explorer.eu/foods See the Phenol Explorer website for more information on sources of precursors.]
* [http://phenol-explorer.eu/classifications/compounds/15/15 Food sources of Hydroxycinnamic acids (p-Coumaric acid, ferulic acid, caffeic acid, etc.).]
* [https://thebrulab.libsyn.com/episode-030-phenolic-compounds-in-beer-w-dr-mike-lentz Brü Lab Podcast Episode 030 | Phenolic Compounds In Beer w/ Dr. Mike Lentz.]
{| class="wikitable sortable"
| 4-Vinylphenol <ref name="Doss">[http://www.ahaconference.org/wp-content/uploads/presentations/2008/GregDoss_BrettBrewing.pdf Doss, Greg]. Brettanomyces:
Flavors and performance of single and multiple strain
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> || C<sub>8</sub>H<sub>8</sub>O <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> || C<sub>9</sub>H<sub>10</sub>O<sub>2</sub>. 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>. || 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>. Ferulic Some studies have found that ferulic acid is generally more efficiently extracted from a combination of 70% barley malt and 30% wheat malt (not raw wheat), despite studies showing that barley malt often contains more ferulic acid than wheat malt (see [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 || || C<sub>8</sub>H<sub>8</sub>O<sub>2</sub> <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>.
====Acid Production====
In the presence of oxygen and alcohol, ''Brettanomyces'' species produce acetic acid as a byproduct of glucose fermentationrespiratory metabolism. The more oxygen that is present, the more acetic acid is produced and the less ethanol is produced by ''Brettanomyces'' <ref>[https://link.springer.com/article/10.1007/s00253-002-1197-z Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. M. G. Aguilar Uscanga, M.L. Délia, P. Strehaiano. 2003.]</ref><ref>[https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain," Escarpment Labs blog post, 01/20/21.]</ref><ref name="Rozpędowska" /><ref>[https://academic.oup.com/femsyr/article/13/1/34/544881?login=true Fernanda Cristina Bezerra Leite, Thiago Olitta Basso, Will de Barros Pita, Andreas Karoly Gombert, Diogo Ardaillon Simões, Marcos Antonio de Morais, Jr, Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants, FEMS Yeast Research, Volume 13, Issue 1, February 2013, Pages 34–43, https://doi.org/10.1111/j.1567-1364.2012.12007.x]</ref><ref>[https://link.springer.com/article/10.1007/BF00400180 Wijsman, M.R., van Dijken, J.P., van Kleeff, B.H.A. et al. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie van Leeuwenhoek 50, 183–192 (1984). https://doi.org/10.1007/BF00400180.]</ref>. In an environment with oxygen present, sugar ''Brettanomyes'' switches to respiratory metabolism. Sugar is reduced to pyruvate within the cell and is then broken down into acetaldehyde which is then enzymatically oxidized into acetic acid or ethanol (dubbed the Custers effect). The acetate that is produced by ''Brettanomyces'' under aerobic conditions is an important requirement for the cells to fully metabolize certain types of sugars like galactose <ref>[https://link.springer.com/article/10.1007/s12010-023-04398-w Teles, G.H., Xavier, M.R., Da Silva, J.M. et al. The Metabolism of Respiring Carbon Sources by Dekkera bruxellensis and Its Relation with the Production of Acetate. Appl Biochem Biotechnol (2023). https://doi.org/10.1007/s12010-023-04398-w.]</ref>. This is thought to be a defensive tactic against competing microorganisms (e.g. ''Brettanomyces'' has been shown to produce more acetic acid when co-fermented with ''S. cerevisiae'', and ''S. cerevisiae'' has been shown to have less viability over time in the presence of acetic acid and ethanol) <ref>[https://link.springer.com/article/10.1023/A:1022592810405 Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH. S.N. FreerB. DienS. Matsuda. 2003.]</ref><ref name="Hubbe"></ref>. Depending on the brewer's palate and the degree of acetic production, this can be a desirable or undesirable trait. The degree of acetic acid production varies among different ''Brettanomyces'' species and strains, and it is limited by limiting oxygen exposure (see [[Mixed_Fermentation#Aging|aging mixed fermentation beer]] for practical tips on limiting oxygen exposure). For example, ''B. naardenensis'' and ''B. custersianus'' produce less acetic acid than other species of ''Brettanomyces'' <ref name="colomer_2020_genome" /><ref name="Tiukova_2019" />. Acetic acid produced by ''Brettanomyces'' is also used in the synthesis of [[Brettanomyces#Ester_Production|acetate esters]] such as ethyl acetate, perhaps as a mechanism to protect itself after hindering other microbes via the acetic acid precursor. ''Brettanomyces'' has been shown is not known 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 significant amounts 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 lactic acid as a byproduct of fermentation under semi-aerobic conditions, but not anaerobic conditions <ref name="Smith_2018" />.
Michael Lentz and Chad Harris tested whether or not the hydroxycinnamic acids (HCAs) inhibit the growth of ''Brettanomyces''. They found that high levels of hydroxycinnamic acids (HCAs), which includes ferulic acid, p-coumaric acid, and caffeic acid, do inhibit the growth of ''Brettanomyces''. Ferulic acid is the strongest inhibitor of these three HCAs with most strains tested not being able to grow in wort that contained 12 mM (millimolar) of ferulic acid. Caffeic acid was generally shown to be the weakest inhibitor of the three HCAs tested. Levels of 25 mM p-coumaric acid inhibited the growth of all strains tested, and levels of 30 mM of caffeic acid inhibited all strains tested. The ability of HCAs to inhibit growth is different from strain to strain of ''Brettanomyces''. Inhibition does not appear to be species dependent. Some strains display a lag time and grow more slowly in the presence of high amounts of HCA's, but still eventually achieve maximum growth compared to if they were grown without exposure to HCAs, while others lag and then stop growing before reaching maximum growth <ref name="Lentz"></ref>.
Pure cultures. In cooperation with [http://www.funkfactorygeuzeria.com/2013/06/brett-strain-guide.html Funk Factory].
===[[Bootleg Biology]]/[[Spot Yeast]]===
{| class="wikitable sortable"
|-
|-
| Lambicus||''Dekkera bruxellensis''||''Brettanomyces bruxellensis''||Brettanomyces lambicus||High intensity Brett character. Know to produce the “horsey” aroma characteristic of Brettanomyces yeast. Classic strain used in secondary fermentation for Belgian style beers and lambics. Same as White Labs. || '''Commercial pitches only.'''
|-
|}
===[[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. ||
|-
|}
|}
===Fermentis==={| class="wikitable sortable"|-! Common Name !! Species Name !! Synonym Name !! Lab/Package !! Flavor/Aroma !! Source Note|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces 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"
|-
|}
===[[GigaYeast]](CLOSED)===
{| class="wikitable sortable"
|-
|}
===[[Mainiacal Yeast]](CLOSED)===
{| class="wikitable sortable"
|-
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || MIP-703 Brett Brux III || A strain used for secondary fermentation in Belgian-style beers such as lambics. Characterized as "fruity with tropical fruit dominating the aroma" <ref name="propagate_website" />. ||
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || YH169 || In collaboration with [[Wild Pitch Yeast]]. High attenuation, produces a lemony/tart/subtle “Brett” aroma and flavors characterized as lemon, limoncello, and mint with a slight undertone of the more typical Brett barnyard funk. Can be used at a wide variety of temperatures (68-99 F) with a potential flavor sweet spot of 80-85 F. Would work well in a saison or Brett IPA <ref name="propagate_website" />. || Isolated from a spontaneous fermentation in Indianapolis, IN.
|-
| Unknown || Unknown || Unknown || MIP-710 Brett Stave I || strain used for secondary fermentation in Belgian-style beers such as lambics; characterized as "fruity". || This particular strain was isolated from a Colorado Brewery and produces intense fruit notes <ref name="propagate_website" />.
|-
| Unknown || Unknown || Unknown || MIP-760 Brett phantom || A highly fruity strain that will ferment well by itself or pitched into a blend. || Isolated from a famous saison producer <ref name="propagate_website" />.
|-
| Unknown || Unknown || Unknown || BTN-70 Feints || Characterized as sweet tarts, ripe peach skin, clementine, white grapes, and a hint of candied strawberry with a backbone of soft Brett funk. || Isolated from a natural wine from Mendocino County, California <ref name="propagate_website" />.
|-
| Unknown || Unknown || Unknown || BTN-81 Yellow Jacket || Characterized as Brett-forward with notes of sweet tarts, ripe peach skin, dried lime peel, tangerine pith. || Isolated from a yellow jacket insect <ref name="propagate_website" />.
|-
|}
Just like in other yeast species, temperature has a direct effect on the rate of growth for ''Brettanomyces''. The optimal growth rate temperature range for ''Brettanomyces'' is between 25-32°C (77-90°F). Growth is about half as slow at 20°C (68°F). ''Brettanomyces'' will still grow at temperatures as low as (and maybe lower than) 15°C (59°F) and will be much slower, however one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth temperature range of 20-32°C. At a temperature of 35°C (95°F), both growth and viability over time are greatly inhibited <ref name="Brandam_2008">[http://oatao.univ-toulouse.fr/1595/1/Brandam_1595.pdf Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects. Brandam C, Castro-Martínez C, Délia ML, Ramón-Portugal F, Strehaiano P. 2008.]</ref>.
* See this [https://www.facebook.com/groups/MilkTheFunk/posts/7060769660617854/ MTF thread] on anecdotes using different types of yeast nutrients.
* For information on mixed culture starters, see [[Mixed_Cultures#Starters_and_Other_Manufacturer_Tips|Mixed Culture Starters]].
Oxygen levels are an important factor to consider when deciding which of the above two methods to use for a ''Brettanomyces'' starter. ''Brettanomyces'' creates acetic acid in the presence of oxygen, potentially leading to higher levels of ethyl acetate, which is considered an off flavor in higher amounts. As the amount of oxygen increases, cell growth increases, but so does acetic acid production. The amount of acetic acid produced is species/strain dependent, so some strains may benefit from more aeration without having the negative effect of creating too much acetic acid. Other strains may need a less aerobic starter (semi-aerobic) in order to produce the highest cell count with minimal acetic acid <ref>[http://www.ncbi.nlm.nih.gov/pubmed/12655458 Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. Aguilar Uscanga, Délia1, and Strehaiano. 2003.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0010(199712)75:4%3C489::AID-JSFA902%3E3.0.CO;2-9/abstract Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking. Maurizio Ciani and Luisa Ferraro. April 1999.]</ref><ref>[http://link.springer.com/article/10.1023%2FA%3A1014927129259 Acetic acid production by Dekkera/Brettanomyces yeasts. S.N. Feer. April 2002.]</ref>. In addition to acetic acid production, it has been observed that some ''Brettanomyces'' strains grown under aerobic conditions continue to produce THP when transferred to anaerobic conditions. See [[Tetrahydropyridine#Brettanomyces|THP]] for details.
This presents a sort of "catch 22" when growing ''Brettanomyces'' in a starter. The brewer must weigh the pros and cons of how much aeration to provide. If the ''Brettanomyces'' is going to be used in a [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]], for example, then a stir plate with foil covering the flask is the best choice. If the ''Brettanomyces'' is instead being pitched in secondary with the intention of long aging, then having a high cell count isn't as necessary and the risk of adding more acetic acid/ethyl acetate to an aging beer is greater. If a lot of acetic acid is produced during the starter, then they can opt to cold crash and decant the starter. ''Brettanomyces'' can have a difficult time flocculating and settling out, even when cold crashed. The brewer may need to allow a few days for the cells to fully sediment <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1099473923414154/?comment_id=1099522943409252&offset=0&total_comments=25&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Richard Preiss of Escarpment Yeast Labs on MTF. 6/26/2015.]</ref>. Additionally, ''Brettanomyces'' that is cold crashed may be slower to begin fermentation. If the brewer believes that the amount of acetic acid produced was insignificant, then cold crashing can be skipped and the entire starter can be pitched. Even if the starter has a lot of acetic acid, the amount of acetic acid in the volume of a starter is fairly insignificant once diluted into a full batch of wort or beer. If the starter is not going to be used within a month, then an aerobic starter is not the best option since the presence of a lot of acetic acid will slowly kill the ''Brettanomyces'' over time. In this case, the starter should be lightly shaken (or occasionally manually stirred), and an airlock put in place on the flask in order to keep out most of the oxygen.
Although more experiments are probably needed, agitation is believed to be an important factor for any species of microbe (yeast and bacteria). Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174865305875015&reply_comment_id=1176092372418975&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.]</ref>.
====Pitching Rate Calculators====
Current yeast pitching calculators for brewers are not adequate for determining ''Brettanomyces'' pitching rates based on starter volume size because the maximum cell density of ''Brettanomyces'' per mL of wort is 3 to 6 times the cell density of ''Saccharomyces''. For example, a given ''Saccharomyces'' strain may reach a cell density of 130 million cells per mL in a 1.040 wort (different ''Saccharomyces'' strains can have different cell densities as well, although they are a lot lower than ''Brettanomyces'' overall). Different ''Brettanomyces'' strain cell densities have been reported to be 600 to 885 million cells per mL in 1.040 wort depending on the species/strain <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>.
Given this information, many brewers historically have been using the lager pitching rate settings in online yeast pitching calculators for ''Brettanomyces'' starters (around 2000 mL for 5 gallons, for example). Effectively, this means they have been pitching around 4 to 5 times the amount of ''Brettanomyces'' cells that they thought they were pitching. However, if this very high pitching rate is giving good results for brewers, it should continue to be used. Exploration of ''Brettanomyces'' pitching rates for 100% Brett fermentations is something to be desired once we know what our pitching rates actually are, and many brewers have been pitching 4-5 times the pitching rate for lagers if they use an online yeast pitching rate calculator instead of counting the cells under a [[Microscope|microscope]].
====[[Escarpment Laboratories]]====
* [https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain For Beer."]
* Presentation by Richard Preiss:
: <youtube>NyGbnDMDn0Q</youtube>
===Pasteurization===
''Brettanomyces'' has complete thermal death at 122°F (50°C) for 5 minutes <ref name="Nunes de Lima 2020" /><ref name="Couto_2005" /> . See also [[Barrel#Sanitizing|Barrel Sanitizing]] and [[Quality_Assurance#Pasteurization|Pasteurization]].
===Catching/Bioprospecting Wild ''Brettanomyces''===
See [[Wild_Yeast_Isolation#Wild_Brettanomyces|Isolating Wild ''Brettanomyces'']].
==See Also==
* [[Brettanomyces secondary fermentation experiment]]
* [[Brettanomyces Storage Survival Experiment]]
* [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]
* [[Crooked Stave Artisan Beer Project]]
* [[Scientific Publications]]