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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, around the same time that the first mammals emerged <ref name="Rozpędowska">[https://www.nature.com/articles/ncomms1305 Rozpędowska, E., Hellborg, L., Ishchuk, O. et al. Parallel evolution of the make–accumulate–consume strategy in Saccharomyces and Dekkera yeasts. Nat Commun 2, 302 (2011). https://doi.org/10.1038/ncomms1305.]</ref>. Both genera evolved independently to ferment sugar and produce ethanol <ref name="Schifferdecker">[http://onlinelibrary.wiley.com/doi/10.1002/yea.3023/pdf The wine and beer yeast Dekkera bruxellensis. Anna Judith Schifferdecker, Sofia Dashko, Olena P. Ishchuk, and Jure Piškur. 7 July 2014.]</ref><ref name="Gounot_2019">[https://www.biorxiv.org/content/10.1101/826990v1.full High complexity and degree of genetic variation in Brettanomyces bruxellensis population. Jean-Sébastien Gounot, Cécile Neuvéglise, Kelle C. Freel, Hugo Devillers, Jure Piškur, Anne Friedrich, Joseph Schacherer. 2019. DOI: https://doi.org/10.1101/826990 .]</ref>. Although first isolated from beer in 1889 by H. Seyffert of the Kalinkin Brewery in St. Petersberg and again in 1899 by scientists J. W. Tullo at Guinness, the discovery of ''Brettanomyces'' was first publicly published by the Director of laboratory of the New Carlsberg Brewery, Hjelte Claussen, in 1904 after he cultured it in 1903 from English beers that exhibited a sluggish secondary fermentation . At the time, he included these newly discovered yeasts in the genus ''Torula'' <refname="Gilliland_1961">[https://crescentcitybrewtalk.com/brettanomyces-i/ "BRETTANOMYCES I OCCURRENCE, CHARACTERISTICS, AND EFFECTS ON BEER FLAVOUR" by R. B. Gilliland, B.A., B.Sc, F.R.I.C. (Arthur Guinness Son & Co. (Dublin) Ltd., St. James’s Gate, Dublin). Received 21st Janurary, 1961.] See also [http://barclayperkins.blogspot.com/2013/06/when-was-brettanomyces-discovererd.html "When was Brettanomyces discovered?" Ron Pattenson. Shut Up About Barclay Perkins blog. 06/29/2013. retrieved Retrieved 08/18/2016.]</ref><ref>[http://breweryhistory.com/journal/archive/149/Yeast.pdf Ray Anderson. "ONE YEAST OR TWO? PURE YEAST AND TOP FERMENTATION". The Brewery History Society. 2012.]</ref>. At the time of discovery, Claussen was aiming to recreate the flavor profile of traditional English ales by fermenting them with pure cultures of ''Saccharomyces'', and either pitching pure cultures of his newly discovered ''Brettanomyces'' yeast along with ''Saccharomyces'', or as he preferred, after the primary fermentation of ''Saccharomyces'' <ref>[https://www.facebook.com/download/448702618652516/GB190328184A.pdf "Improvements in and connected with the Manufacture of English Beers or Malt Liquors and in the Production of Pure Yeast Cultures for use therein." Patent application by Hjelte Claussen for ''Brettanomyces''. A.D. 1903.]</ref>. Although Claussen saw the character from ''Brettanomyces'' as a desirable character in ales and , along with [[Hops#The_Freshening_Power_of_the_Hop_.28Hop_Creep.29|dry hop creep]], was identified its character as a quality the source of traditional English secondary fermentation during long aged ales, at some point ''Brettanomyces'' became identified as a contaminate in wineries contributing to their lasting high carbonation <ref>[https://archive.org/details/principlespracti00syke "The principles and breweries due to some practice of the phenolsbrewing" Sykes, acidsWalter John. London, and haze that it sometimes producesC. These phenols Griffin and acids have generally been described as "barnyard"Company, "burnt plastic"limited, "wet animal", "fecal", and "horse sweat", although some tasters describe these flavors with different terminology because they perceive certain flavor compounds differently while some other tasters simply cannot detect certain flavor compounds at all 1907. Pgs 384-388.]<ref name="smith_divol_2016" /><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]</ref><ref>[https://www. Lucy Joseph, Elizabeth Ayoutube. Albinocom/watch?v=9BwO7gbhdns Martyn Cornell interview on Craft Beer Channel, Susan E"The Time Is Now – reinventing the English IPA". Ebeler, Linda F. Bisson. 201509/28/2022.]</ref>. The general viewpoint of brewers (other than Lambic and Flanders red/brown brewers 8 minutes in Belgium) and vintners . Beer historian, [https://barclayperkins.blogspot.com/search?q=brettanomyces Ron Pattinson], has stated that ''Brettanomyces'' is primarily a spoilage organism held for many decades, and still holds in most cases. More recently the positive flavor components that have been identified was typically present in 1800''Brettanomyces'' beer s English aged beers such as pineapplestock ales, pale ales, stone fruitsporters, and barrel aged IPA's that were shipped to some degree acetic acidIndia, have regained popularity with brewers outside and it was considered an important component of Belgium. Wine tasters have also described wines with certain both the flavor compounds derived profile of these beers and in protecting beer from contaminants via ''Brettanomyces'' as positive characteristics fermenting the majority of wineresidual sugars <ref>[https://www.crowdcast.io/e/IPA-Past-Present-Future/1 Ron Pattinson. It is important to keep in mind that individual tasters on tasting panels describe some flavor compounds as "negative" while others describe them as "positive" (and sometimes a mixed response is given by a taster in regards to a certain flavor compound), and this discrepancy in interpretations History of ''Brettanomyces'' derived flavor compounds appears IPA -1700s to be based on personal preference and experience2021". In some cases, for example for vinyl phenols, low levels that are not detectable by some people, but detected by others contribute positively to wine, while higher amounts contribute negativelyDoug Piper's interview with Ron Pattinson. Thus, a lower intensity of some flavor compounds can be seen as more desirable07/25/2021. Overall, the enjoyment or displeasure of the various flavor compounds produced by ''Brettanomyces'' and at certain levels is completely subjective ]</ref name="Lucy_2015" />(~56 and 59 mins in )<ref>[https://www.guildsommfacebook.com/public_contentmilkthefunkthepodcast/featuresvideos/articles/b/kelli-white/posts/brettanomyces Brettanomyces: Science & Context. Kelli White. GuildSomm website1097016944410369 Ron Pattinson. 03/29/2018MTF Live. Retrieved 0502/10/20192022.]</ref>(45 minutes in).
Following the discovery of this yeast by Claussen, isolates of ''Brettanomyces'' were discovered in Belgian lambic beers in the 1920's. At this time, ''Brettanomyces'' was proposed as the new genus name, separating them from the genus ''Torula'' <ref name="Gilliland_1961" />. The species name 'bruxellensis', meaning 'Brussels' in Latin, became the proposed species name for ''B. bruxellensis.''. This yeast species was then isolated from other industrial fermentations such as wine, cider, kombucha, kefir, olives, and bioethanol production. Custers was the first to attempt to describe the rest of the genus using phenotypic characteristics in 1940. In 1960, J. van der Walt observed some species of ''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 desirable character 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 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'' are considered synonyms have recently been genetically analyzed and reclassified as strains of ''B. bruxellensis'' <ref name="Agnolucci_2017">[https://link.springer.com/article/10.1007/s11274-017-2345-z Brettanomyces bruxellensis yeasts: impact on wine and winemaking. Monica Agnolucci, Antonio Tirelli, Luca Cocolin, Annita Toffanin. 2017.]</ref>. Of these five species, only ''B. anomalus'' and ''B. bruxellensis'' have been identified to have a teleomorph version. In their teleomorph version they are referred to as ''Dekkera anomala'' and ''Dekkera bruxellensis'' <ref name="smith_divol_2016"></ref><ref name="Schifferdecker"></ref><ref name="Steensels"></ref><ref name="Crauwels_2014">[http://aem.asm.org/content/80/14/4398.full Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Sam Crauwels, Bo Zhu, Jan Steensels, Pieter Busschaert, Gorik De Samblanx, Kathleen Marchald, Kris A. Willems, Kevin J. Verstrepen and Bart Lieven. 2014.]</ref>. All of the other names such as the ones often used by yeast labs (e.g. "claussenii") are derived from old nomenclature that is no longer used scientifically ([http://www.sciencedirect.com/science/article/pii/S0168160515001865#t0005 click here] for a table that lists old and new taxonomical nomenclature). Most ''Brettanomyces'' cultures from brewer's yeast labs are classified genetically as ''B. bruxellensis'' or ''B. anomalus''.
Recently a new species of ''Brettanomyces'' has been proposed, although classification has not been fully established. The proposed name is ''Brettanomyces acidodurans'' sp. nov. Two strains of ''B. acidodurans'' were isolated from olive oil from Spain and Israel; however, its presence in olive oil has been described as "rare" because only two strains were found after searching dozens of olive oils. Its closest relation is to ''B. naardenesis'' by 73% of its genetic makeup. No teleomorph form was observed. This species is a strong acetic acid producer, and it is very tolerant of acetic acid in its environment. It can consume lactose and cellobiose but does not consume maltose. it is unknown but a possibility that this species contributes to the vinegary taste of spoiled olive oils, although this has generally been attributed to acetic acid bacteria <ref>[https://www.ncbi.nlm.nih.gov/pubmed/28160110 Brettanomyces acidodurans sp. nov., a new acetic acid producing yeast species from olive oil. Péter G, Dlauchy D, Tóbiás A, Fülöp L, Podgoršek M, Čadež N. 2017.]</ref>.
A genetic survey of 145 different strains of ''B. bruxellensis'' from 29 countries, 5 continents, and 9 different fermentation niches was conducted in 2018 by Avramova et al. They found that these strains formed roughly 6 genetic groups with mostly separate ancestral lineages, and 1 group with a mixed ancestral lineage: 3 wine groups, 1 beer group, 1 kombucha group (most distantly related to the beer group), as well as 1 tequila/ethanol group that has multiple ancestral lineages <ref name="Avramova_2018" />. These groups are partially determined by the identification of at least two hybridization events that happened during the evolution of ''B. bruxellensis'', similar to the hybridization events that created the Saaz and Frohberg subgroups of ''S. pastorianus'' (the parents of these hybridization events in ''B. bruxellensis'', whether from different species or not, has yet to be determined and will require whole genome sequencing of species closely related to ''B. bruxellensis'') <ref name="Gounot_2019" />. This was expressed mostly in the ploidy level of each group (the number of sets of chromosomes), with 2 of the wine groups, the tequila group, and the beer group containing more sets of chromosome pairs than the other groups (diploid vs triploid; this is thought to encourage adaption and hybridization). Additionally, the triploid wine group was generally more tolerant of SO<sup>2</sup> than the diploid wine groups <ref name="Avramova_2018" />. [https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637 Colomer et al (2020)] surveyed the whole genomes of 64 strains of ''B. bruxellensis'', 14 strains of ''B. anomalus'', 3 strains of ''B. custersianus'', and 3 strains of ''B. naardenensis''. They broke the ''B. bruxellensis'' beer group into two clades: a "farmhouse" clade which comprised of strains of ''B. bruxellensis'' isolated from commercial craft beer breweries, and a "lambic" clade which comprised of strains isolated from spontaneously fermented Belgian lambic beers. There was also a subdivision of the lambic clade which comprised of strains of ''B. bruxellensis'' identified with various natural origins; ethanol plants, barrel-aged beers, and matured wines. This subclade was called "wild/wood" by the researchers. See also[https:* //www.frontiersin.org/files/Articles/495404/fmicb-11-00637-HTML-r1/image_m/fmicb-11-00637-g002.jpg Figure 2 (family tree)] from the study <ref name="colomer_2020_genome">[https://www.facebookfrontiersin.org/articles/10.3389/fmicb.2020.com00637/groupsfull Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications. Marc Serra Colomer, Anna Chailyan, Ross T. Fennessy, Kim Friis Olsson, Lea Johnsen, Natalia Solodovnikova and Jochen Forster. 2020. DOI: https:/MilkTheFunk/permalinkdoi.org/336469455355873510.3389/ A collection fmicb.2020.00637.]</ref>. Science has also begun to explore targeted gene manipulation of ''B. bruxellensis'' via CRISPR, which will eventually lead to a better understanding of early 1900the ''Brettanomyces bruxellensis''s papers by Claussen genome <ref>[https://link.springer.com/article/10.1007/s00253-020-10750-5 Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system. Cristian Varela, Caroline Bartel, Cristobal Onetto, Anthony Borneman. Applied Microbiology and Alfred Chapman; MTF post by Cory WidmayerBiotechnology. 2020.]</ref>.
===Morphology===
* [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]].
''Brettanomyces'' is known for not producing much glycerol commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries (although the median occurrence of ''Brettanomyces'' in barrels may be very low to none within a given winery or brewery depending on their hygiene and other factors <ref>[https://link.springer.com/article/10.1007/s00217-011-1523-8 Guzzon, R., Widmann, G., Malacarne, M. et al. Survey of the yeast population inside wine barrels and the effects of certain techniques in beerpreventing microbiological spoilage. Eur Food Res Technol 233, 285–291 (2011). https://doi.org/10.1007/s00217-011-1523-8.]</ref><ref>[https://enagris.wikipediafao.org/wikiagris-search/Glycerol Glycerol] is a colorlesssearch.do?recordID=IT2007601151 Fontanot, S.; Ninino, sweet-tastingM.E.; Comi, viscous liquid that is thought to be an important contributor to G.; Elimination of Dekkera/Brettanomyces from barriques of the mouthfeel Italian CDO Isonzo area. Controlled Designation of beerOrigin; Friuli-Venezia Giulia. 2006.]</ref>). Glycerol is produced These include structures such as a stress response by a wide range wooden fermentation vessels, walls of microbesthe building, including as well as the inside surface of wood barrels and actually buried within the wood of barrels. ''S. cerevisiaeBrettanomyces''has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and various species occasionally as deep as 5 to 8 mm, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and strains of one study found that the ''DebaryomycesBrettanomyces''was able to penetrate the French oak barrels up to 8 mm, while only penetrate American oak barrels up to 4 mm) of the barrel <ref name="Agnolucci_2017" /><ref name="Cartwright_2018">[http://www.ajevonline.org/content/early/2018/05/23/ajev.2018.18024 Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam. Zachary M. Cartwright, Dean A. Glawe, Charles G. Edwards. 2018. DOI: 10.5344/ajev.2018.18024.]</ref>, with the highest concentration of surviving cells being at the top staves where oxygen is more accessible (although Cartwright et al. found the opposite was true, perhaps due to methodology of sampling or a difference in 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 ''CandidaBrettanomyces''that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of ''LachanceaBrettanomyces'', and on wine grapes. Liquid ozone has been shown to be less effective at eliminating ''ZygosaccharomycesBrettanomyces''. Despite not producing amounts Heating the inside of glycerol that are perceivable in beer, some strains the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing ''Brettanomyces bruxellensis'' actually produce glycerol which is stored inside within the wood of their cells as a response to osmotic stress. They can also uptake glycerol into their cells. Doing so allows the cells to survive osmotic pressure barrels (see [[Barrel#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.sciencedirectncbi.nlm.nih.com/sciencegov/articlepubmed/pii/S0740002013001251?via%3Dihub Osmotic stress response in the 25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensisby hot water treatment. Silvia GalafassiFabrizio, Marco ToscanoVigentini, Ileana VigentinParisi, Jure PiškurPicozzi, Concetta Compagno, Foschino. 20132015.]</ref><ref>[https://academicwww.oupsciencedirect.com/femslescience/advance-article-abstract/doipii/10.1093/femsle/fny020/4828327?redirectedFrom=fulltext Osmotolerance S1466856417310068 Control of Dekkera Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and the role of two Stl glycerol-proton symportersgaseous ozone. Jana ZemančíkováFrancesco Craveroa, Michala DuškováVasileios Englezos, Hana ElicharováKalliopi Rantsiou, Klára PapouškováFabrizio Torchio, Hana SychrováSimone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. It is currently not known how many strains are capable Although the role of producing glycerol internally''Brettanomyces'' appears to be limited in distillation, or if this amount it has been isolated during the fermentation process of glycerol tequila making. It has any impact on perceived mouthfeel also been isolated from drains, pumps, transfer hoses, and other equipment that is difficult to sanitize. The survivability of ''Brettanomyces'' has also partly been attributed to its ability to form a beer if [[Quality_Assurance#Biofilms|biofilm]] (in particular ''B. bruxellensis''). Microorganisms that can form a substantial amount of biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. ''Brettanomyces'' cells eventually autolyze (see [https://wwwhas therefore been identified as a significant contaminate for breweries and wineries.facebook Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''B.com/groups/MilkTheFunk/permalink/2003626776332193/ this MTF thread])bruxellensis''. The role of glycerol ''Brettanomyces'' is also commonly found in creating mouthfeel sherry, and is debatable found (although only rarely) in olive production, lemonade, kombucha, yogurt, pickles, and soft drinks. ''B. anomalus'' and ''B. bruxellensis'' are generally found much more commonly than the wine world other three species of ''Brettanomyces'' <refname="smith_divol_2016">[httpshttp://www.winesandvinessciencedirect.com/featuresscience/article/68760 Tim Pattersonpii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. "Many Roads to Mouthfeel"Brendan D. Wines & Vines MagazineSmith, Benoit Divol. Nov 2009. Retrieved 03/23/2018June 2016.]</ref>.
See also:
* [http://www.foodsci.purdue.edu/research/labs/enology/Winemaking%20Calculations%20Spring%20Workshop%204_29_2011.pdf?fbclid=IwAR3WnDfo6k9hqfo4EvnWrp4GwFiB7qHoexifJBfLnIEIhLwRtC2Hm7qkUug Calculations for SO<sub>2</sub> additions] and [https://www.facebook.com/groups/592560317438853/?comment_tracking=%7B%22tn%22%3A%22R2%22%7D associated MTF discussion].
* [[Quality_Assurance#VBNC_In_Yeast|Viable But Nonculturable Yeast.]]
====Biofilm====
[[File:Chlamydospore Brett.JPG|thumb|First evidence of possible (unconfirmed <ref name="heit_lebleux">[https://www.facebook.com/groups/MilkTheFunk/permalink/3118617694833090/?comment_id=3120943487933844 Dr. Bryan Heit. Milk The Funk Facebook group thread on Lebleux et al. (2019) and chlamydospore in Brettanomyces. 12/11/2019.]</ref>) chlamydospore cell structures of ''B. bruxellensis'', found in a biofilm. Photo by [https://www.sciencedirect.com/science/article/abs/pii/S0168160519303952 Lebleux et al. (2019)] <ref name="Lebleux_2019">[https://www.sciencedirect.com/science/article/abs/pii/S0168160519303952 New advances on the Brettanomyces bruxellensis biofilm mode of life. Manon Lebleux, Hany Abdo, Christian Coelho, Louise Basmaciyan, Warren Albertin, Julie Maupeu, Julie Laurent, Chloé Roullier-Gall, Hervé Alexandre, Michèle Guilloux-Benatier, Stéphanie Weidmann, Sandrine Rousseaux. 2019. DOI: https://doi.org/10.1016/j.ijfoodmicro.2019.108464.]</ref>.]]
''Brettanomyces'' has the ability to form a [[Quality_Assurance#Biofilms|biofilm]]. Biofilm formation is a survival mechanism induced by stress whereby the cells adhere to non-living surfaces such as plastic and stainless steel<ref>[https://ives-technicalreviews.eu/article/view/4544 "Brettanomyces bruxellensis biofilms: a mode of life to withstand environmental stresses?" Sandrine Rousseaux, Manon Lebleux, Hany Abdo, Louise Basmacyian, Chloé Roullier-Gall, Hervé Alexandre, Stéphanie Weidmann. 2020. DOI: https://doi.org/10.20870/IVES-TR.2020.4544.]</ref>. After adhesion to the surface, the cells produce a protective layer of proteins and polysaccharides that help protect the organism from cleaning and sanitizing agents. Joseph et al. (2007) tested 36 wine strains of ''B. bruxellensis'' for biofilm formation over a 10 day period. They found that just under half of the strains formed a biofilm, and about half of those formed considerable and consistent biofilms throughout the tests. Almost all strains tested (95%) adhered to a surface with 0.1% glucose within 6 hours of contact (the same conditions that get ''Saccharomyces cerevisiae'' to adhere to a surface; longer contact with surfaces and higher residual sugar could encourage ''Brettanomyces'' to adhere more readily to surfaces). A juice-based growth media in the range range of 2 - 4.5 pH was tested for biofilm formation and 3-4 for cell adhesion to a surface, and for most of the strains they formed stronger biofilms and adhered better in the higher pH growth media (4.5 pH being the highest tested). Under a pH of 3.5 significantly dropped biofilm formation and adherence, indicating that something about pH affects the cells ability to attach themselves. The researchers concluded that winemakers should keep wine in the lower end of the pH range (3.5). Six different types of cleaners were tested to see how well they removed the biofilms: keytones + surfactant detergent, quaternary ammonia + surfactant detergent, sodium hydroxide (caustic soda), sodium carbonate (soda ash), sodium hydroxide + surfactant (alkaline detergent), and chlorine (sanitizer, not a detergent). They found that only caustic soda was consistently efficient at removing the biofilm. The chlorine, while it did not remove the biofilm, still killed all of the ''Brettanomyces'' cells, and it was presumed that the other cleaners might have killed the ''Brettanomyces'', but that was not tested for. They also tested to see if cells that were adhered to a surface could be cleaned. Again, the caustic soda performed consistently the best, but the ammonia + surfactant cleaner and the quaternary ammonia + surfactant detergent also effectively removed adhered cells. The other cleaners varied in how well they removed adhered cells from a surface <ref>[https://www.researchgate.net/publication/235411588_Adhesion_and_biofilm_production_by_wine_isolates_of_Brettanomyces_bruxellensis Adhesion and biofilm production by wine isolates of Brettanomyces bruxellensis. C. M. Lucy Joseph, Gagandeep Renuka Kumar, Gagandeep Renuka Kumar, Edward Su, Linda F Bisson. 2006. American Journal of Enology and Viticulture 58(3):373-378.]</ref>.
Dimopoulou et al. (2019) studied ''Brettanomyces bruxellensis'' biofilms from each of the genetic branches of ''B. bruxellensis''. They found that for the wine strains biofilms formed more readily when grown in wine must rather than YPD media; however, the beer strains grew biofilms equally well in wine must and YPD media. The biofilms contained a large portion of saturated fatty acids and a smaller portion of monounsatured fatty acids. The amount of exopolysaccharide produced varied widely across the strains tested per cell population, with some wine strains producing little EPS (40 mg/L/OD), beer strains producing moderate amounts, and the other wine group producing a high amount (100 mg/L/OD). Additionally, the different strains displayed a varying degree of negative cell wall charges, with the beer and tequila strains being more negatively charged than wine strains, which could help them adhere to surfaces and form biofilm <ref>[https://europepmc.org/article/PPR/PPR73221?singleResult=true Dimopoulou M., Renault M., Dols-Lafargue M., Albertin-Leguay W., Herry J., Bellon-Fontaine M., Masneuf-Pomarede I. 2019. DOI: 10.1101/579144.]</ref>.
* [[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==
''Brettanomyces'' strains may possess both alpha and beta-glucosidases. Beta-glucosidase is intracellular (works on sugars that are passed into the cell through the cell wall), while alpha-glucosidase is both intracellular and extracellular (released into the environment by the cell). <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref><ref name="Kumara_1993">[http://aem.asm.org/content/59/8/2352.short Localization and Characterization of α-Glucosidase Activity in Brettanomyces lambicus. H. M. C. Shantha Kumara, S. De Cort and H. Verachtert. 1993.]</ref> These enzymes allow ''Brettanomyces'' strains to break down a broad range of sugars, including long-chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to ''Saccharomyces'' yeasts. <ref name="Steensels"></ref><ref>[http://www.scribd.com/doc/277758178/Insight-into-the-Dekkera-anomala-YV396-genome Insight into the Dekkera anomala YV396 genome. Samuel Aeschlimann. Self-published on Eureka Brewing Blog. Spet 2015.]</ref>.
Extracellular and intracellular alpha-glucosidase activity has been shown to break down sugars up to 9-12 chain carbons in one strain of ''B. lambicus'' (now classified as ''B. bruxellensis''), which is partly responsible for the slow, over-attenuation of wort that some strains of ''Brettanomyces'' an achieve in beers such as lambic and American sour beers <ref name="yakobson_introduction"></ref><ref name="smith_divol_2016"></ref>. Alpha-glucosidases are the enzymes that allow them to break down maltose, turanose, melezitose, and trehalose, as well as dextrins such as maltotetraose and maltopentaose. These enzymes work by cleaving off glucose that can be directly consumed by the cell, leaving a shorter chain sugar behind which is then further broken down. In the case of extracellular alpha-glucosidase activity, this breakdown of complex sugars occurs outside of the cell and may benefit other microorganisms if present such as lactic acid bacteria. These dextrins are left over after a normal ''Saccharomyces'' fermentation <ref name="Steensels"></ref>. Some other polysaccharides can be fermented by ''Brettanomyces'', including starch, laminarin, and pectin <ref name="Crauwels1"></ref>. The more complex the starch or sugar, the slower it is hydrolyzed by the alpha-glucosidase enzymes. The optimal pH for the alpha-glucosidase enzyme produced by one strain of ''B. bruxellensis'' was 6 and at a temperature of 39-40°C (102-104°F), and its activity was greatly reduced below a pH of 4.5 and above 8 (although citric acid was used as a buffer, and its effects on the enzyme was not compared to other acids), which might contribute to slower ''Brettanomyces'' fermentation in acidic beers <ref name="Kumara_1993" />. A survey of 84 strains from several species of ''Brettanomyces'' showed that there is a wide variability in the ability of different strains to ferment maltose, with some strains not being able to ferment it at all and others fermenting it very slowly, suggesting that alpha-glucosidase is not functional or poor in some strains. Additionally, when maltose is present instead of just glucose, the researchers saw an increased lag during the growth phase <ref name="colomer_2020_genome" />.
''B. bruxellensis'' and ''B. nanus'' can also produce oligo-1,6-glucosidase, which hydrolyze the alpha-1,6 linkages in starch and glycogen to produce oligosaccharides, and then further break down these oligosaccharides to produce sugars with alpha-1,4 linkages (for example, maltose, in the case of starches from malted barley <ref>[https://www.ncbi.nlm.nih.gov/books/NBK22396/ Complex Carbohydrates Are Formed by Linkage of Monosaccharides. Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. 2002.]</ref>). These alpha-1,4 linkages (maltose) are then further broken down by the maltose enzyme by strains of ''Brettanomyces'' or other present microbes that produce this enzyme <ref name="Crauwels_2014" />. Unlike for some domesticated diastatic ''S. cerevisiae'' strains, this ability for ''Brettanomyces'' to break down starches has occurred in the wild without domestication <ref name="roach_2019" /><ref>"Amylases". H. Taniguchi, Y. Honnda, in Encyclopedia of Microbiology (Third Edition), 2009.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3049768678384659/ Ben Sykes. Milk The Funk Facebook post on ''Brettanomyces'' evolution to ferment dextrins. 11/13/2019.]</ref>.
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.] See also: [https://www.facebook.com/groups/MilkTheFunk/permalink/1285391951489016/ "Funky can be Great: Brettanomyces bruxellensis Beer Fermentations" (poster for study). Caroline Tyrawa, Richard Preiss, and George van der Merwe. 2017.]</ref>. Colomer et al. (2020) surveyed the whole genome of 64 strains of ''B. bruxellensis'' and found that beer strains are more likely to be able to ferment maltose (alpha-glucosidase) but not cellobiose (beta-glucosidase) and wine/wild strains tend to have the opposite tendency, indicating that ''B. bruxellensis'' strains have adapted to the environments in which they live over time <ref name="colomer_2020_genome" />.
Currently, research into how well ''Brettanomyces'' strains ferment the trisaccharide maltotriose has not been explored much by science. However, 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):
Colomer et al. (2020) was the first to examine the effects of ''Brettanomyces'' on hops during fermentation. The researchers selected 4 strains of ''B. bruxellensis'' and 1 strain of ''B. anomalus'' that had the genetic markers for producing beta-glucosidase enzyme, and fermented them as a primary fermenter in a non-dry hopped beer, and also performed a second experiment where they inoculated the ''Brettanomyces'' strains in a dry hopped beer that was first fermented with ''S. cerevisiae''. Monoterpene alcohols were measured before and after inoculation with the 5 strains of ''Brettanomyces''. In both beers, they found a decrease in geraniol and a rise in beta-citronellol after the inoculation with ''Brettanomyces''. Beta-citranellol reached a level of 31.5 μg/L with one of the strains, which is a much higher level of beta-citronellol than anything that has been reported with ''S. cerevisiae'', suggesting that some strains of ''Brettanomyces'' might be better at converting monoterpenes from hops than ''Saccharomyces''. See [https://onlinelibrary.wiley.com/doi/full/10.1002/jib.610 Figure 5] in the open access study. Interestingly, the strains with the highest beta-glucosidase activity produced the lowest amount of beta-citranellol, indicating that there is no link between beta-glucosidase activity in ''Brettanomyces'' and hop biotransformation. This might be due to the fact that most of the beta-glucosidase enzyme is produced within the cell and is not released outside of it. The researchers hypothesized that this conversion could be due to two proteins referred to as BbHye2 and BbHye3 that can be present in ''Brettanomyces'' metabolism <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.610 Biotransformation of hop derived compounds by Brettanomyces yeast strains. Marc Serra Colomer, Birgitte Funch, Natalia Solodovnikova, Timothy John Hobley, Jochen Förster. 2020. DOI: https://doi.org/10.1002/jib.610.]</ref>.
For more information on glycosides, see the [[Glycosides]] page. For more infomration information on hop biotransformation in general, see the [[Hops#Hop_Derived_Compounds_In_Beer_and_Biotransformations|Hops]] page.
===Nitrogen Metabolism===
''Brettanomyces'' is capable of synthesizing several ethyl esters from ethanol and fatty acids, as well as other types of esters from various alcohol types (methanol, for example). Among the most prolific of these are ethyl acetate (synthesized from ethanol and acetic acid), ethyl lactate (synthesized from ethanol and lactic acid), phenethyl acetate, ethyl caproate, ethyl caprylate, ethyl deconoate <ref name="Tyrawa_2017" />, along with the hydrolysis (breakdown) of isoamyl acetate. Esters have been found to attract fruit flies and other flying insects, which help many species of yeast transfer from one food source to another (namely 2-phenyl-ethanol, 3-methyl-1-butanol, ethyl acetate, 2-methyl-1-butanol, and 3-methyl-3-butenol). Some of these esters are also released by blooming flowers and it is thought that the attraction to flowers by insects is also driven by these same esters <ref>[https://onlinelibrary.wiley.com/doi/epdf/10.1002/ece3.3905 Chemical signaling and insect attraction is a conserved trait in yeasts. Paul G. Becher, Arne Hagman, Vasiliki Verschut, Amrita Chakraborty, Elżbieta Rozpędowska, Sébastien Lebreton, Marie Bengtsson, Gerhard Flick, Peter Witzgall, Jure Piškur. 2018.]</ref>. During non-mixed fermentations where lactic acid is minimal to none, insignificant amounts of ethyl lactate esters are produced, whereas ethyl caprylate and ethyl caproate have a general increase. With the addition of lactic acid, ethyl lactate levels are greatly increased although may still not reach the flavor threshold level of 250 mg/L (strain dependent), and ethyl acetate is generally slightly increased. The amounts of esters produced vary widely based on species and strain <ref>[http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad]. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Production of Secondary Metabolites. 2011.</ref>. A similar but slower evolution of esters has been seen in a long-term study on examining how Belgian lambic from Cantillon ages in bottles. The study found that lactic acid (produced by lactic acid bacteria) and ethyl lactate increased as bottles aged, while ethyl decanoate and isoamyl acetate decreased, all presumably from ''Brettanomyces'' metabolism over time <ref>[http://horscategoriebrewing.blogspot.com/2016/02/thoughts-on-spitaels-and-van.html "Thoughts on Spitaels and Van Kerrebroeck et al, 2015." Dave Janssen. Hors Catégorie Blog. 02/20/2016. Retrieved 03/15/2016.]</ref>.
Ester production peaks towards the end of growth and is influenced by temperature, aeration/agitation, and pH. Spaepen and Verachtert found in one study that the optimal temperature for growth and thus ester production was 28°C (77°F), although they did not test higher temperatures. This study also found that continuously shaken samples produced relatively fewer esters, as well as samples that were not exposed to oxygen at all. The highest ester production was found under conditions of limited oxygen supply(semi-aerobic versus aerobic or anaerobic), no agitation, held at a temperature of 28°C (77°F), and young cells produced more esters than older cells. It also found that esterase activity (esterase is the enzyme that facilitates ester production and destruction) increases as pH rises until a pH of 7.6 is reached, after which it begins to decline again. It was shown that the ester formation/degradation was indeed caused by enzymatic activity of any ''Brettanomyces'' species/strain, and not caused by chemical reactions or from ''Saccharomyces'' or ''Kloeckera'' activity <ref name="Spaepen"></ref>. Another study by Tyrawa et al. found that all strains of ''B. bruxellensis'' tested produced above threshold levels of ethyl caproate, ethyl caprylate, and ethyl deconoate esters at 15°C versus 22.5°C, but for some strains the higher fermentation temperature of 22.5°C produced significantly more of these esters than the lower 15°C temperature (other strains produced similar levels of esters at both temperatures, although they fermented slower at 15°C) <ref name="Tyrawa_2017" />.
Pitching rate of ''Brettanomyces'' may have a slight effect on ester production levels, but the differences caused by pitching rate probably do not have a significant impact on the sensory character of the beer <ref name="MTF_Brett_Secondary"></ref>. ''Brettanomyces'' produces higher levels of esters when fermented without competition from ''S. cerevisiae'', and this correlates with higher ''Brettanomyces'' cell growth when not in competition with ''S. cerevisiae'' (see [[100%25_Brettanomyces_Fermentation#Are_100.25_Brett_Beers_Really_Cleaner.3F|100% ''Brettanomyces'' Fermentation]]) <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/permalink/1407620509266159/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. The aromatic amino acids phenylalanine, tryptophan, and tyrosine have been associated with higher ester formation <ref name="Lucy_2015" />.
High levels of bicarbonate can affect the ester production of ''B. bruxellensis'', as well as the production of acids and phenols. One study reported that levels of 100 mg/l produced significantly higher ethyl acetate, but there was less of an effect on other esters. High amounts of bicarbonate over 100 mg/l in the 100% ''B. bruxellensis'' fermentations produced significantly lower amounts organic acids (hexanoic, octanoic, and decanoic acid) and lower amounts of vinyl phenols <ref name="Thompson-Witrick_2022" />. See also The Brü Lab Podcast with [http://thebrulab.libsyn.com/episode-087-impact-bicarbonates-have-on-brettanomyces-fermentations-w-dr-katherine-thompson-witrick Dr. Thompson-Witrick].
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>. Almost 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>[https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637/full Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications. Marc Serra Colomer, Anna Chailyan, Ross T. Fennessy, Kim Friis Olsson, Lea Johnsen, Natalia Solodovnikova and Jochen Forster. 2020. DOI: https://doi.orgname="colomer_2020_genome" /10.3389/fmicb.2020.00637.]</ref>.
These vinyl derivatives have similar tastes to the ethyl derivatives but have lower flavor thresholds. Levels of all compounds produced vary depending on species and strain of ''Brettanomyces''. Although the production of ethyl phenols has been identified to occur higher in substrates with more available sugars, and this has also correlated with higher growth <ref name="Barata_2008">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00415.x/full The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine. André Barata, Daniela Pagliara, Tiziana Piccininno, Francesco Tarantino, Wilma Ciardulli, Manuel Malfeito-Ferreira, Virgílio Loureiro. 2008. DOI: 10.1111/j.1567-1364.2008.00415.x]</ref>, some data supports that pitching rate does not have an effect on how much phenol content is produced by ''Brettanomyces''<ref name="MTF_Brett_Secondary">[http://www.milkthefunk.com/wiki/Brettanomyces_secondary_fermentation_experiment Brettanomyces secondary fermentation experiment. Milk The Funk Wiki. Lance Shaner and Richard Preiss. Retrieved 04/21/2016.]</ref>. Additionally, Curtin et al. (2013) showed that while both cell growth and attenuation was inhibited in anaerobic conditions in wine, phenol production was not (in fact, the phenol production was inhibited by aerobic conditions). They also showed that each of the three strains of ''B. bruxellensis'' tested all produced the same amount of phenols, while other flavor compounds such as esters were produced at different levels by each of the strains <ref>[https://www.ncbi.nlm.nih.gov/pubmed/24010603 Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma. Curtin CD, Langhans G, Henschke PA, Grbin PR. 2013]</ref>. [https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert's Bachelors thesis] also reported no correlation between fermentation rate and phenol production in several strains of ''B. bruxellensis'' <ref name=Humbert_2021">[https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert for the degree of Honors Baccalaureate of Science in Chemical Engineering presented on May 21, 2021. Title: Performance of Brettanomyces Yeast Strains in Primary and Secondary Beer Fermentations.]</ref>. Perhaps growth itself is not as much of a factor in producing phenols, but having sugars available for metabolism is. This contradicts the somewhat popular belief that under-pitching ''Brettanomyces'' produces more "funky" flavors. Additionally, perhaps some strains are perceived as "funkier" than others due to less ester production and more fatty acid production (isobutyric acid, for example), rather than more phenol production.
Another study by Tyrawa et al. found that fermentation temperature also did not have a significant effect on phenol production in 9 strains of ''B. bruxellensis''. Given the same wort composition, strains of ''B. bruxellensis'' produced similar levels of phenols at both 15°C and 22.5°C. The ester production was affected by this temperature difference in some strains but not others (see [[Brettanomyces#Ester_Production|Esters]] above). Assuming that phenols contribute the "funky" flavor characteristics, this suggests that perhaps a lower balance of esters to phenols produces a more "funky" tasting beer more so than a beer with more phenol content. If so, a lower fermentation temperature may be one way to emphasize phenols over fruity esters <ref name="Tyrawa_2017" />. Both Tyrawa and Humbert reported that there was no correlation between flavor profiles from phenol production of different strains of ''Brettanomyces bruxellensis'' and their origin <ref name=Humbert_2021" />.
It has been hypothesized that the production and destruction of various phenols by ''Brettanomyces'' is connected with the [https://en.wikipedia.org/wiki/Redox redox balance]; however, this has not been demonstrated. Ethyl phenols have been shown to be highly attractive to fruit flies, and it has also been proposed that these aromatics allow ''Brettanomyces'' to travel from food source to food source and by doing so increasing its chances of survival in the wild <ref>[http://www.sciencedirect.com/science/article/pii/S0960982214016558 Olfaction: Smells Like Fly Food. Geraldine A. Wright. 2015.]</ref><ref name="smith_divol_2016"></ref>. Phenols have been shown to have positive effects on decreasing protein glycation, a complication associated with type 2 diabetes <ref>[http://www.asbcnet.org/publications/journal/vol/2017/Pages/ASBCJ-2017-1323-01.aspx High Phenolic Beer Inhibits Protein Glycation In Vitro. Susan M. Elrod, Phillip Greenspan, Erik H. Hofmeister. 2017. ]</ref>.
* [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>.
|-
| 4-Ethylphenol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Barnyard, Horsey, Spicy, Smoky, Medicinal, Band-Aid <ref>[http://www.aroxa.com/wine/wine-flavour-standard/4-ethyl-phenol/ Aroxa Website. 4-Ethyl Phenol. Retrieved 08/19/2015.]</ref>) || Ethyl phenol || 4-vinylphenol || 0.3 ppm (odor; in beer) <ref name="Maarse">[https://books.google.com/books?id=_OvXjhLUz-oC&pg=PA513&lpg=PA513&dq=4-ethylphenol+odor+threshold&source=bl&ots=fzhA9yvvrJ&sig=K0QykyRqj9TnezG1Mih4gLru1ZE&hl=en&sa=X&ved=0CC4Q6AEwAmoVChMI77DfhNi1xwIV0jqICh1zVgQ8#v=onepage&q=4-ethylphenol%20odor%20threshold&f=false Volatile Compounds in Foods and Beverages. Henk Maarse. CRC Press, Mar 29, 1991. Pg 514, 515.]</ref> || C<sub>8</sub>H<sub>10</sub>O <ref name="pubchem_4EP">[http://pubchem.ncbi.nlm.nih.gov/compound/4-ethylphenol PubChem Website. 4-Ethylphenol. Retrieved 08/19/2015.]</ref> || Also known as 1-Ethyl-4-hydroxybenzene and P-Ethylphenol <ref name="pubchem_4EP"></ref>. Identified as a major product of ''B. bruxellensis'' <ref name="Lucy_2015" />. Richard Preiss of [[Escarpment Laboratories]] describes pure 4EP as the following, "barnyardy with a slight solvent edge at low concentrations, and full on hospital antiseptic/bandaid/barnyard at high concentrations. Really quite complex, but maybe not in a good way." <ref name="Preiss_4EP_4EG">[https://www.facebook.com/groups/MilkTheFunk/permalink/2141048572590012/?comment_id=2141122099249326&reply_comment_id=2141606422534227&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss. Milk The Funk Facebook group thread on the flavor of 4EP and 4EG. 06/22/2018.]</ref>
====Acid Production====
In the presence of oxygen, ''Brettanomyces'' species produce acetic acid as a byproduct of glucose fermentationrespiratory metabolism. The more oxygen that is present, the more acetic acid is produced and the less ethanol is produced by ''Brettanomyces'' <ref>[https://link.springer.com/article/10.1007/s00253-002-1197-z Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. M. G. Aguilar Uscanga, M.L. Délia, P. Strehaiano. 2003.]</ref><ref>[https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain," Escarpment Labs blog post, 01/20/21.]</ref><ref name="Rozpędowska" /><ref>[https://academic.oup.com/femsyr/article/13/1/34/544881?login=true Fernanda Cristina Bezerra Leite, Thiago Olitta Basso, Will de Barros Pita, Andreas Karoly Gombert, Diogo Ardaillon Simões, Marcos Antonio de Morais, Jr, Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants, FEMS Yeast Research, Volume 13, Issue 1, February 2013, Pages 34–43, https://doi.org/10.1111/j.1567-1364.2012.12007.x]</ref><ref>[https://link.springer.com/article/10.1007/BF00400180 Wijsman, M.R., van Dijken, J.P., van Kleeff, B.H.A. et al. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie van Leeuwenhoek 50, 183–192 (1984). https://doi.org/10.1007/BF00400180.]</ref>. In an environment with oxygen present, 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>.
* [http://www.horscategoriebrewing.com/2019/01/spontaneous-fermentation-and-biogenic.html "Spontaneous fermentation and biogenic amines" by Dr. Dave Janssen; review of several studies that looked at the levels of biogenic amines in different beers and their production, and their potential flavor contribution.]
* [http://suigenerisbrewing.com/index.php/2019/01/22/biogenic-amines/ "Fact or Fiction – Biogenic Amines in Beer" by Dr. Bryan Heit; an analysis of biogenic amines in spontaneously fermented beer and associated health concerns.]
====Glycerol====
''Brettanomyces'' is known for not producing much glycerol in beer. [https://en.wikipedia.org/wiki/Glycerol Glycerol] is a colorless, sweet-tasting, viscous liquid that is thought to be an important contributor to the mouthfeel of beer. Glycerol is produced as a stress response by a wide range of microbes, including ''S. cerevisiae'', and various species and strains of ''Debaryomyces'', ''Candida'', ''Lachancea'', and ''Zygosaccharomyces''. Despite not producing amounts of glycerol that are perceivable in beer, some strains of ''Brettanomyces bruxellensis'' actually produce glycerol which is stored inside of their cells as a response to osmotic stress. They can also uptake glycerol into their cells. Doing so allows the cells to survive osmotic pressure <ref>[https://www.sciencedirect.com/science/article/pii/S0740002013001251?via%3Dihub Osmotic stress response in the wine yeast Dekkera bruxellensis. Silvia Galafassi, Marco Toscano, Ileana Vigentin, Jure Piškur, Concetta Compagno. 2013.]</ref><ref>[https://academic.oup.com/femsle/advance-article-abstract/doi/10.1093/femsle/fny020/4828327?redirectedFrom=fulltext Osmotolerance of Dekkera bruxellensis and the role of two Stl glycerol-proton symporters. Jana Zemančíková, Michala Dušková, Hana Elicharová, Klára Papoušková, Hana Sychrová. 2018.]</ref>. It is currently not known how many strains are capable of producing glycerol internally, or if this amount of glycerol has any impact on perceived mouthfeel of a beer if a substantial amount of ''Brettanomyces'' cells eventually autolyze (see [https://www.facebook.com/groups/MilkTheFunk/permalink/2003626776332193/ this MTF thread]). The role of glycerol in creating mouthfeel is debatable in the wine world <ref>[https://www.winesandvines.com/features/article/68760 Tim Patterson. "Many Roads to Mouthfeel". Wines & Vines Magazine. Nov 2009. Retrieved 03/23/2018.]</ref>.
====Other Compounds====
|-
| Tetrahydropyridine (Cheerios®, mousy, urine, cracker biscuit, corn chips) || Ketone <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1034461653248715/?comment_id=1034611563233724&offset=0&total_comments=25 Humbard, Matt. Milk The Funk Discussion. 3/10/2015.]</ref> || L-Lysine, ethanol, and oxygen || Varies || See the ''[[Tetrahydropyridine]]'' page for more details.
|-
| Hydrogen sulfide (rotten egg) || Chalcogen hydride gas <ref>[https://en.wikipedia.org/wiki/Hydrogen_sulfide "Hydrogen sulfide". Wikipedia. Retrieved 08/29/2020.]</ref> || || 4 µg/l in beer <ref>[https://www.aroxa.com/beer/beer-flavour-standard/hydrogen-sulphide/ "Hydrogen sulphide". Aroxa. Retrieved 08/29/2020.]</ref> || Produced in high amounts by ''B. custersianus'' and ''B. naardenensis'' <ref name="colomer_2020_genome" />.
|-
| Diacetyl/butanedione (butter) || Vicinal diketone <ref>[https://en.wikipedia.org/wiki/Diacetyl "Diacetyl". Wikipedia website. Retrieved 08/29/2020.]</ref> || || 0.1–0.2 ppm in lager and 0.1–0.4 ppm in ales, although flavor thresholds as low as 17 ppb and 14–61 ppb have been reported <ref name="krogerus_2013">[https://www.researchgate.net/publication/259331290_125th_Anniversary_Review_Diacetyl_and_its_control_during_brewery_fermentation Krogerus, K. and Gibson, B.R. (2013), 125th Anniversary Review: Diacetyl and its control during brewery fermentation. J. Inst. Brew., 119: 86-97. https://doi.org/10.1002/jib.84.]</ref>. || Produced by all strains and all species of ''Brettanomyces'' except ''B. naardenensis''; the amount that it is produced varies widely and not much is known about what determines diacetyl levels produced from ''Brettanomyces'' <ref name="colomer_2020_genome" />. ''Brettanomyces'' generally produce very low amounts of diacetyl (0.019 - 0.048 mg/L). It is hypothesized that ''Brettanomyces'' can reduce diacetyl during its maturation phase in a similar way to ''Saccharomyces'' species, but this has not been investigated that we are aware of. It has been reported that diacetyl reduction is faster at a lower pH of around 3.5, which is a typical pH range for sour beer and might be one of the contributing factors to a lack of anecdotal reports of diacetyl in sour beer <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.381 Michel, M., Meier‐Dörnberg, T., Jacob, F., Methner, F. ‐J., Wagner, R. S., and Hutzler, M. (2016) Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications. J. Inst. Brew., 122: 569– 587. doi: 10.1002/jib.381.]</ref><ref name="krogerus_2013" />.
|-
|}
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. ||
|-
|}
===[[Escarpment Laboratories]]===
* [https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain," a guide to Escarpment Laboratories ''Brettanomyces'' products on their blog.]
{| class="wikitable sortable"
|-
! Common Name !! Species Name !! Synonym Name !! Lab/Package !! Flavor/Aroma !! Source Note
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || Brett B || A classic Brettanomyces bruxellensis strain, typically used in secondary fermentations. Attenuation: 70-85% // Optimum Temp: 26ºC+ (80F+) // Alcohol tolerance: Medium-high // Flocculation: Low <ref name="escarpment_strains">[http://www.escarpmentlabs.com/strains "Strains" list. Escarpment Laboratories website. Retrieved 12/07/2017.]</ref>. ||Isolated from a bottle of Orval <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1278416635519881/?comment_id=1279065935454951&reply_comment_id=3665429823485205 Richard Preiss. Milk The Funk Facebook group post about Escarpment Labs Brett B culture.. 07/11/2020.]</ref>.
|-
| Claussenii || ''Dekkera anomala'' || ''Brettanomyces anomalus'' || Brett C || A strain of Brettanomyces clausenii, now understood to be among the Brettanomyces anomalus species. Minimal funk, tends to exhibit fruity pineapple or mango notes. Pairs well with fruit and/or hops. Recommended for secondary or co-fermentation as attenuation is variable. Attenuation: Highly Variable // Optimum Temp: 18-25ºC (64.4-77F) // Flocculation: Medium-low. Potentially no longer available (no longer listed on website) <ref name="escarpment_strains" />. ||
|}
===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"
|-
|}
===[[Jasper Yeast LLC]]===
{| class="wikitable sortable"
|-
! Common Name !! Species Name !! Synonym Name !! Lab/Package !! Flavor/Aroma !! Source Note
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY033 - Brett brux Amsterdam || Slow growing, good for conditioning in the style of Orval || Amsterdam, Netherlands <ref name="jasper_brett"Chateaux>[https://jasperyeast.com/yeast/brettanomyces "Brettanomyces". Jasper Yeast Website.]</ref>|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY044 - Brett brux Rosa || Comparable to JY033, but slightly more funky, tart and aggressive. || California, USA <ref name="jasper_brett" />|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY87 JY061 - Brett brux Chateaux || JY87 is a Brettanomyces yeast isolated by Jasper Yeast LLC. Fast-growing, it has almost Saccharomyces-like doubling times. Shows great fermentation as primary strain in a variety of beers. Ferments fast to 60% attenuation, after which fermentation slows down and more flavor and aroma is produced. Strong pineapple and stonefruit aroma after prolonged fermentations (3-9 months). Great companion to beers that could use some funk, and complements hoppy beers perfectly. Flocculation is low, strain will form a pellicle when oxygen is present. Sequencing of ITS regions indicated Brettanomyces bruxellensis. Micrograph of JY87 cells coming soon. || West-Flanders, Belgium.<ref name="jasper_brett"/>|-| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY062 - Brett brux Abbey || Mild aromatics and phenolics, but quite tart and high tendency to produce acetic acid in the presence of oxygen. || California, USA <ref name="jasper_brett"/>|-| Lambicus || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || JY157 - Brett brux lambicus || Good for Brettanomyces beers where an intense character is preferred. Prolonged aging can produce almost a cherry like sourness. Optimum temperature: 80-90°F (27-32°C) || Belgium <ref name="jasper_brett"/>|-| Claussenii || ''Dekkera anomala'' || ''Brettanomyces anomalus'' || JY196 - Brett c || Optimum temperature: 80-90°F (27-32°C). Pineapple aroma, but can also develop a leather character. This yeast is not suitable to be used on its own in malt based worts because of its limited fermentation capacity. || Belgium <ref name="jasper_brett"/>|-
|}
===[[Mainiacal Yeast]](CLOSED)===
{| class="wikitable sortable"
|-
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || Brettanomyces bruxellensis OYL-216 || White wine character and light funk, and develops its character rather quickly. Brett character will be apparent within a few weeks of reaching terminal gravity and will continue to develop if given additional conditioning time. Flocculation: low, Attenuation: 70-85%, Temp: 68-80°F, Alcohol Tolerance: medium-high. Potentially the same ''B. bruxellensis'' strain as their C2C blend <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3345112128850311/?comment_id=3345153185512872 Aaron Kester and Mark Schwarz. Milk The Funk Facebook thread about OYL-216. 03/14/2020.]</ref>. Pro brewers only. || A US Northwest brewery.
|-
|}
===[[Propagate Lab]]===
{| class="wikitable sortable"
|-
! Common Name !! Species Name !! Synonym Name !! Lab/Package !! Flavor/Aroma !! Source Note
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || MIP-701 Brett Brux I || Used for secondary fermentation in Belgian-style beers such as lambics, this strain creates a medium-intensity, earth-forward character in finished beer. A historic brewery in Belgium uses this yeast in secondary fermentation and bottling to produce the signature flavor of its beer <ref name="propagate_website">[http://www.propagatelab.com Propagate website. Retrieved 06/20/2020.]</ref>. || (Editor's note: this is likely to be a strain isolated from Orval).
|-
| Bruxellensis || ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' || MIP-702 Brett Brux II || a strain used for secondary fermentation in Belgian-style beers such as lambics. It creates a medium-intensity, earth-forward character in finished beer. Balanced between barnyard and fruit <ref name="propagate_website" />. ||
|-
| 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-714 Brett Stave IV || Produces barnyard like aromas and is a very aggressive strain. || This strain was isolated from a bottle of beer originally produced in the Netherlands <ref name="propagate_website" />.
|-
| Clausenii || ''Dekkera anomala'' || ''Brettanomyces anomalus'' || MIP-720 Brett clausenii || A strain used for secondary fermentation in Belgian-style ales and English Old Ales. The yeast can be fairly neutral in aroma and works well by itself. When it is paired with a phenol producing yeast it will create barnyard like aromas <ref name="propagate_website" />. ||
|-
| Unknown || Unknown || Unknown || MIP-750 Brett tool || An aggressive strain that produces strong barnyard aroma. Works well as a blend. || Isolated from a bottle of European beer fermented with fruit <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" />.
|-
|}
|-
| [[East Coast Yeast]] || ECY34 Dirty Dozen Brett Blend || Twelve (12) different isolates of Brettanomyces exhibiting high production of barnyard "funk" and esters. Dryness, ripe fruit, and acidity will be encountered over a period of months and over time (>1 yr), may display gueuze-like qualities in complexity. Contains various isolates from lambic-producers, B. bruxellensis, B. anomalus, B. lambicus, and B. naardenensis. For those who want the most from Brett yeast, whether a 100% Brett fermentation is desired or adding to secondary aging projects. Suggested fermentation temperature: 60-74 F. Attenuation high. [https://www.facebook.com/groups/MilkTheFunk/permalink/1258857067475838/ See this MTF thread for fermentation tips for 100% and mixed fermentations].
|-
| [[Escarpment Laboratories]] || Berliner Brett Blend || A blend of Berliner Brett 1 (''Brettanomyces anomalus'') and Berliner Brett 2 (''Brettanomyces bruxellensis'') for balanced, subtle Brett character in traditional Berliner Weisse and beyond. Flavour profile includes citrus, white wine, and peach. Secondary pitch rates only.
|-
| [[Escarpment Laboratories]] || MOTHERSHIP Brett Blend || This blend typically contains 10 individual strains. The character is highly dependent on fermentation conditions, but tends toward balanced, medium to high intensity Brett character.
|-
| [[Fermmento Labs]] (Brazil) || FB1 Tropical funky Bugs || Contains ''B. custersianus'' and 'B. anomalus''.
|-
| [[Mainiacal Yeast]] || Hurricane Brett Blend || Mix of all 70 ''Brettanomyces'' strains at Mainiacal Yeast. Commercial brewery pitches always available, and occasionally available to homebrewers <ref name="Amaral_Mainiacal" />.
|-
| [[Propagate Lab]] || MIP-765 Native Brett Blend || Blend of ''Brettanomyces'' isolated from a number of natural sources. Isolates are from plums, natural wines, wild plants, and spontaneous fermentation. This blend will add complexity to any beer <ref name="propagate_website" />.
|-
| [[The Yeast Bay]] || Beersel || Not overly funky but there is a sweaty note hanging behind lemon and citrus fruits, nice blend of subtle funk and citrus/fruit. All strains were identified as ''B. bruxellensis'' <ref name="TYB_Blends">[https://www.facebook.com/groups/MilkTheFunk/permalink/1178127178882161/?comment_id=1178188512209361&offset=0&total_comments=9&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Nick Impellitteri regarding taxonomy of the TYB Brett blends. 11/13/2015.]</ref>. Making a starter is fine despite the instructions advising against it on the vial and will not greatly effect the character of the final beer <ref name="TYB_starter_is_ok">[https://www.facebook.com/groups/MilkTheFunk/permalink/1551348668226675/?comment_id=1551707504857458&reply_comment_id=1551745278187014&comment_tracking=%7B%22tn%22%3A%22R%2313%22%7D Conversation on MTF with Nick Nick Impellitteri from The Yeast Bay. 01/16/2017.]</ref>.
===Starter Information===
When pitching just ''Brettanomyces'' from a commercial pure or blended culture and no other microbes, it is recommended to make a starter for the culture. If the ''Brettanomyces'' is being pitched into secondary, no starter is necessary unless the brewer suspects that the ''Brettanomyces'' has lost a lot of viability due to age, heat exposure, etc., or prefers higher cell count pitches (current information suggests that there is no significant flavor difference between high and low pitching rates in secondary pitches of ''Brettanomyces''; see [[Brettanomyces secondary fermentation experiment]]).
Starter wort made with dried malt extract at around 1.040 starting gravity is adequate for most strains of ''B. bruxellensis''. For ''Brettanomyces'' strains such as many ''B. anomalus'' strains that don't ferment maltose, a mixture of 50% DME and 50% table sugar or dextrose at 1.040 starting gravity should be adequate. Yeast nutrient at the manufacturer's recommended rate can be added if sufficient growth is not observed <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/4208440909184091/?comment_id=4209205899107592 Dr. Bryan Heit. Milk The Funk Facebook thread post on starters for ''B. anomalus''. 01/07/2021.]</ref>.
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]].
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1585625541465654/ MTF thread on The Yeast Bay Brett/Sacch flavor profiles.]
====[[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]]
* [http://byo.com/stories/issue/item/262-brettanomyces "Brettanomyces", Steve Piatz in Brew Your Own Magazine, October 2005.]
* [http://www.horscategoriebrewing.com/2017/06/100-year-old-czech-beer.html "100 year old Czech beer," by Dave Janssen (musings on the presence of ''Brettanomyces'' and high amounts of acetic and lactic acid in a 100 year old lager).]
* [https://www.youtube.com/watch?v=qaX7tiFQ2iA "Brewing with Brettanomyces Yeast and Mixed Cultures" presentation by Chad Yakobson at BC Craft Brewers Guild, 2019.]
==References==