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==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>. ''Brettanomyces'', along with [[Hops#The_Freshening_Power_of_the_Hop_.28Hop_Creep.29|dry hop creep]], was identified as the source of secondary fermentation during long aged ales, contributing to their lasting high carbonation <ref>[https://archive.org/details/principlespracti00syke "The principles and practice of brewing" Sykes, Walter John. London, C. Griffin and Company, limited, 1907. Pgs 384-388.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/4709953772366133 Gareth Young. Milk The Funk Facebook group thread about English brewers historically relying on Brettanomyces and dry hop creep for carbonation in long aged ales. 06/17/2021.]</ref><ref>[https://www.youtube.com/watch?v=9BwO7gbhdns Martyn Cornell interview on Craft Beer Channel, "The Time Is Now – reinventing the English IPA". 09/28/2022.]</ref>(8 minutes in). Beer historian, [https://barclayperkins.blogspot.com/search?q=brettanomyces Ron Pattinson], has stated that ''Brettanomyces'' was typically present in 1800's English aged beers such as stock ales, pale ales, porters, and barrel aged IPA's that were shipped to India, and it was considered an important component of both the flavor profile of these beers and in protecting beer from contaminants via ''Brettanomyces'' fermenting the majority of residual sugars <ref>[https://www.crowdcast.io/e/IPA-Past-Present-Future/1 Ron Pattinson. "History of IPA -1700s to 2021". Doug Piper's interview with Ron Pattinson. 07/25/2021.]</ref> (~56 and 59 mins in )<ref>[https://www.facebook.com/milkthefunkthepodcast/videos/1097016944410369 Ron Pattinson. MTF Live. 02/10/2022.]</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:
''Brettanomyces'' is commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries (although the median occurrence of ''Brettanomyces'' in barrels may be very low to none within a given winery or brewery depending on their hygiene and other factors <ref>[https://link.springer.com/article/10.1007/s00217-011-1523-8 Guzzon, R., Widmann, G., Malacarne, M. et al. Survey of the yeast population inside wine barrels and the effects of certain techniques in preventing microbiological spoilage. Eur Food Res Technol 233, 285–291 (2011). https://doi.org/10.1007/s00217-011-1523-8.]</ref><ref>[https://agris.fao.org/agris-search/search.do?recordID=IT2007601151 Fontanot, S.; Ninino, M.E.; Comi, G.; Elimination of Dekkera/Brettanomyces from barriques of the Italian CDO Isonzo area. Controlled Designation of Origin; Friuli-Venezia Giulia. 2006.]</ref>). These include structures such as wooden fermentation vessels, walls of the building, as well as the inside surface of wood barrels and actually buried within the wood of barrels. ''Brettanomyces'' has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and occasionally as deep as 5 to 8 mm, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and one study found that the ''Brettanomyces'' was able to penetrate the French oak barrels up to 8 mm, while only penetrate American oak barrels up to 4 mm) of the barrel <ref name="Agnolucci_2017" /><ref name="Cartwright_2018">[http://www.ajevonline.org/content/early/2018/05/23/ajev.2018.18024 Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam. Zachary M. Cartwright, Dean A. Glawe, Charles G. Edwards. 2018. DOI: 10.5344/ajev.2018.18024.]</ref>, with the highest concentration of surviving cells being at the top staves where oxygen is more accessible (although Cartwright et al. found the opposite was true, perhaps due to methodology of sampling or a difference in SO<sub>2</sub> concentrations). Some strains are able to utilize the cellulose of the wood as a carbon source, and occasionally form pseudohyphae within the wood which expands the surface area of the cells allowing them more access to nutrients and allowing them to survive in nutrient deficient environments <ref name="Cartwright_2018" />. Ozone gas has been shown to be an effective way to kill ''Brettanomyces'' that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of ''Brettanomyces'' on wine grapes. Liquid ozone has been shown to be less effective at eliminating ''Brettanomyces''. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing ''Brettanomyces'' within the wood of barrels (see [[Barrel#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment. Fabrizio, Vigentini, Parisi,Picozzi, Compagno, Foschino. 2015.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1466856417310068 Control of Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and gaseous ozone. Francesco Craveroa, Vasileios Englezos, Kalliopi Rantsiou, Fabrizio Torchio, Simone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. Although the role of ''Brettanomyces'' appears to be limited in distillation, it has been isolated during the fermentation process of tequila making. It has also been isolated from drains, pumps, transfer hoses, and other equipment that is difficult to sanitize. The survivability of ''Brettanomyces'' has also partly been attributed to its ability to form a [[Quality_Assurance#Biofilms|biofilm]] (in particular ''B. bruxellensis''). Microorganisms that can form a biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. ''Brettanomyces'' has therefore been identified as a significant contaminate for breweries and wineries. Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''B. bruxellensis''. ''Brettanomyces'' is also commonly found in sherry, and is found (although only rarely) in olive production, lemonade, kombucha, yogurt, pickles, and soft drinks. ''B. anomalus'' and ''B. bruxellensis'' are generally found much more commonly than the other three species of ''Brettanomyces'' <ref name="smith_divol_2016">[http://www.sciencedirect.com/science/article/pii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Brendan D. Smith, Benoit Divol. June 2016.]</ref>.
Unlike most genera of yeast, ''Brettanomyces'' has the characteristics of being very tolerant to harsh conditions, including high amounts of alcohol (up to 14.5-15% ABV <ref name="Crauwels1" /><ref name="Agnolucci_2017" />), a pH as low as 2 <ref>[http://www.winesandvines.com/template.cfm?section=news&content=141954 Wines and Vines. New Research on Role of Yeast in Winemaking; report on a presentation by David Mills and Lucy Joseph from UC Davis. 11/14/2014. Retrieved 08/16/2015.]</ref>, and environments with low nitrogen <ref name="Schifferdecker"></ref> and low sugar sources <ref name="Smith_2018">[https://www.sciencedirect.com/science/article/pii/S0740002017308249 The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium. Brendan D.Smith and Benoit Divol. 2018. DOI: https://doi.org/10.1016/j.fm.2017.12.011.]</ref>. It has been reported that ''B. bruxellensis'' is more tolerant of high levels of bicarbonate than compared to ''S. cerevisiae'' (levels above 100 mg/l slow the fermentation of ''B. bruxellensis'', but do not completely inhibit it, with up to 400 mg/l being tested in one study) <ref name="Thompson-Witrick_2022">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2021.1940654 Katherine A. Thompson-Witrick & Eric R. Pitts (2022) Bicarbonate Inhibition and Its Impact on Brettanomyces bruxellensis Ability to Produce Flavor Compounds, Journal of the American Society of Brewing Chemists, 80:3, 270-278, DOI: 10.1080/03610470.2021.1940654.]</ref>. It has been reported that some strains require a very low concentration of fermentable sugars (less than 300 mg/L) and nitrogen (less than 6 mg/L), which is less than most wines contain <ref name="Smith_2017">[https://www.sciencedirect.com/science/article/pii/S0740002017308249 The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium. Brendan D. Smith, Benoit Divol. 2017.]</ref>. Some strains are able to utilize ethanol, glycerol, acetic acid, and malic acid when no other sugar sources are available <ref name="Smith_2018" />. This capability allows ''Brettanomyces'' to survive in alcoholic beverages such as beer, wine, and cider. In alcoholic beverages, ''B. bruxellensis'' tends to lag after the primary fermentation with ''Saccharomyces''. It is believed that during this lag phase, ''B. bruxellensis'' adapts to the harsh conditions of the beverage (low pH, high concentrations of ethanol, and limited sugar/nitrogen sources). After this lag phase, ''B. bruxellensis'' can grow and survive when no other yeasts can. ''Brettanomyces'' is also more resistant to pH and temperature changes, and tolerant of environments limited in oxygen (although ''Brettanomyces'' prefers the availability of at least a little bit of oxygen). Scientifically, which specific nitrogen and carbon sources ''B. bruxellensis'' uses in these stressful environments has not received much research <ref name="smith_divol_2016"></ref>. [https://www.winesandvines.com/news/article/200000/New-Tools-to-Limit-Wine-Spoilage One study from Dr. Charles Edwards] found that a combination of keeping wine under 54°F (12.2°C) and alcohol at or above 14% resulted in a decline of ''B. bruxellensis'' populations for up to 100 days for two strains that were tested. The study found that neither of the strains grew well at 14% and stopped growth completely at 16% ABV in wine, but one strain grew better than the other at 15%, demonstrating the genetic diversity of ''Brettanomyces''. The researchers concluded that a combination of high ethanol and cold temperatures as well as sulfur dioxide, chitosan, and filtration could be used to control ''Brettanomyces'' in winemaking. ''Brettanomyces'' has been found to be able to grow at temperatures as low as 50°F (10°C) and as high as 95°F (35°C); see [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|fermentation temperature]] for more information <ref>[http://www.ajevonline.org/content/early/2017/01/05/ajev.2017.16102 Interactions between Storage Temperature and Ethanol that Affect Growth of Brettanomyces bruxellensis in Merlot Wine. Taylor A. Oswald, Charles G. Edwards. 2017.]</ref>. ''Brettanomyces'' is also tolerant of IBU's, and there is some evidence that ''Brettanomyces'' is only inhibited by very high IBU's. One study reported that one strain of ''B. bruxellensis'' was inhibited by exposure to 250 mg/L of isomerized hop extract (roughly 250 IBU). Very little inhibition occurred at 150 IBU and about a third of the cells were inhibited at 200 IBU. The inhibited cells were recoverable in YPD media treated with catalase enzyme. In comparison, ''S. cerevisiae'' can be inhibited by 500 mg/L of iso-alpha acids <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2022.902110/full "Transcriptome Analysis of Viable but Non-Culturable Brettanomyces bruxellensis Induced by Hop Bitter Acids". He Yang, Zhao Junfeng, Yin Hua, Deng Yuan. Frontiers in Microbiology. 2022. DOI: 10.3389/fmicb.2022.902110 .] See also [https://www.facebook.com/groups/MilkTheFunk/posts/7473091549385661/ this MTF post]</ref>.
The genetic diversity of ''Brettanomyces'' is particularly wide. For example, one study that analyzed the whole genomes of 53 strains of ''B. bruxellensis'' found that the overall genetic diversity between different strains of ''B. bruxellensis'' was higher than strains of ''S. cerevisiae'' (however, the entire gene set, known as the ''pangenome'', of all the genes among all of the strains of ''B. bruxellensis'' is much smaller than the entire gene set of ''S. cerevisiae'') <ref name="Gounot_2019" />. Some studies have indicated that strains of ''B. bruxellensis'' have adapted to specific environments. For example, one study found that strains of ''B. bruxellensis'' isolated from wine had 20 genes involved in the metabolism of carbon and nitrogen, whereas strains isolated from beer did not. This indicated that ''B. bruxellensis'' strains living in wine have adapted to the harsher environment of wine <ref name="smith_divol_2016"></ref>. Another study found that one out of the two strains tested that were isolated from soda could not ferment maltose, and only strains isolated from wine were able to grow in wine and the beer/soda strains did not. The wine strains were also more resistant to sulfites, which are commonly used in the wine industry to prevent microbial contamination <ref name="Crauwels_2016" />. The whole genome sequencing of one strain of ''B. naardenensis'' and lambic strains of ''B. bruxellensis'' found that they are missing the genes associated with nitrate utilization, indicating that the assimilation of nitrates is not required to survive in beer, perhaps because of the abundance of nitrogen from other sources found in beer <ref name="Tiukova_2019" /><ref name="colomer_2020_genome" />.
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.]
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>.
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| 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>. 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" />. Ferulic acid is also There is also a correlation between how dark a malt is (or how highly kilned it is and how much melanoidin content it has) and how much ferulic acid the malt has; : the darker the malt, the more ferulic acid (however, roasted malts were not tested in the referenced study) <ref>[https://www.mdpi.com/2076-3921/10/7/1124 Shopska V, Denkova-Kostova R, Dzhivoderova-Zarcheva M, Teneva D, Denev P, Kostov G. Comparative Study on Phenolic Content and Antioxidant Activity of Different Malt Types. Antioxidants. 2021; 10(7):1124. https://doi.org/10.3390/antiox10071124.]</ref>. Ferulic acid is stable through the wort boiling process <ref name="kalb_2021" />.
|-
| 4-Vinylcatechol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Plastic, Bitter, Smokey) || Vinyl phenol || Caffeic Acid || || C<sub>8</sub>H<sub>8</sub>O<sub>2</sub> <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/441226 PubChem. 3-Vinylcatechol. Retrieved 08/18/2015.]</ref> || Production level is difference across species/strains of ''Brettanomyces'' <ref name="Oelofse"></ref>.
====Acid Production====
In the presence of oxygen, ''Brettanomyces'' species produce acetic acid as a byproduct of respiratory metabolism. The more oxygen that is present, the more acetic acid is produced and the less ethanol is produced by ''Brettanomyces'' <ref>[https://link.springer.com/article/10.1007/s00253-002-1197-z Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. M. G. Aguilar Uscanga, M.L. Délia, P. Strehaiano. 2003.]</ref><ref>[https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain," Escarpment Labs blog post, 01/20/21.]</ref><ref name="Rozpędowska" /><ref>[https://academic.oup.com/femsyr/article/13/1/34/544881?login=true Fernanda Cristina Bezerra Leite, Thiago Olitta Basso, Will de Barros Pita, Andreas Karoly Gombert, Diogo Ardaillon Simões, Marcos Antonio de Morais, Jr, Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants, FEMS Yeast Research, Volume 13, Issue 1, February 2013, Pages 34–43, https://doi.org/10.1111/j.1567-1364.2012.12007.x]</ref><ref>[https://link.springer.com/article/10.1007/BF00400180 Wijsman, M.R., van Dijken, J.P., van Kleeff, B.H.A. et al. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie van Leeuwenhoek 50, 183–192 (1984). https://doi.org/10.1007/BF00400180.]</ref>. In an environment with oxygen present, ''Brettanomyes'' switches to respiratory metabolism. Sugar is reduced to pyruvate within the cell and is then broken down into acetaldehyde which is then enzymatically oxidized into acetic acid or ethanol (dubbed the Custers effect). The acetate that is produced by ''Brettanomyces'' under aerobic conditions is an important requirement for the cells to fully metabolize certain types of sugars like galactose <ref>[https://link.springer.com/article/10.1007/s12010-023-04398-w Teles, G.H., Xavier, M.R., Da Silva, J.M. et al. The Metabolism of Respiring Carbon Sources by Dekkera bruxellensis and Its Relation with the Production of Acetate. Appl Biochem Biotechnol (2023). https://doi.org/10.1007/s12010-023-04398-w.]</ref>. This is thought to be a defensive tactic against competing microorganisms (e.g. ''Brettanomyces'' has been shown to produce more acetic acid when co-fermented with ''S. cerevisiae'', and ''S. cerevisiae'' has been shown to have less viability over time in the presence of acetic acid and ethanol) <ref>[https://link.springer.com/article/10.1023/A:1022592810405 Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH. S.N. FreerB. DienS. Matsuda. 2003.]</ref><ref name="Hubbe"></ref>. Depending on the brewer's palate and the degree of acetic production, this can be a desirable or undesirable trait. The degree of acetic acid production varies among different ''Brettanomyces'' species and strains, and it is limited by limiting oxygen exposure (see [[Mixed_Fermentation#Aging|aging mixed fermentation beer]] for practical tips on limiting oxygen exposure). For example, ''B. naardenensis'' and ''B. custersianus'' produce less acetic acid than other species of ''Brettanomyces'' <ref name="colomer_2020_genome" /><ref name="Tiukova_2019" />. Acetic acid produced by ''Brettanomyces'' is also used in the synthesis of [[Brettanomyces#Ester_Production|acetate esters]] such as ethyl acetate, perhaps as a mechanism to protect itself after hindering other microbes via the acetic acid precursor. ''Brettanomyces'' is not known to produce significant amounts of lactic acid.
''Brettanomyces'' has been shown to produce enough fatty acids in anaerobic fermentation to drop the pH to 4.0, which can also be esterified (see the ester table above) <ref name="yakobson1"></ref>. Many of these acids can have an unpleasant rancid odor and/or taste, which may be noticeable in young ''Brettanomyces'' beers before these acids are esterified. Some strains can also produce succinic acid as a byproduct of fermentation under semi-aerobic conditions, but not anaerobic conditions <ref name="Smith_2018" />.
! 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).
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===[[GigaYeast]](CLOSED)===
{| class="wikitable sortable"
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Just like in other yeast species, temperature has a direct effect on the rate of growth for ''Brettanomyces''. The optimal growth rate temperature range for ''Brettanomyces'' is between 25-32°C (77-90°F). Growth is about half as slow at 20°C (68°F). ''Brettanomyces'' will still grow at temperatures as low as (and maybe lower than) 15°C (59°F) and will be much slower, however one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth temperature range of 20-32°C. At a temperature of 35°C (95°F), both growth and viability over time are greatly inhibited <ref name="Brandam_2008">[http://oatao.univ-toulouse.fr/1595/1/Brandam_1595.pdf Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects. Brandam C, Castro-Martínez C, Délia ML, Ramón-Portugal F, Strehaiano P. 2008.]</ref>.
* See this [https://www.facebook.com/groups/MilkTheFunk/posts/7060769660617854/ MTF thread] on anecdotes using different types of yeast nutrients.
* For information on mixed culture starters, see [[Mixed_Cultures#Starters_and_Other_Manufacturer_Tips|Mixed Culture Starters]].
Oxygen levels are an important factor to consider when deciding which of the above two methods to use for a ''Brettanomyces'' starter. ''Brettanomyces'' creates acetic acid in the presence of oxygen, potentially leading to higher levels of ethyl acetate, which is considered an off flavor in higher amounts. As the amount of oxygen increases, cell growth increases, but so does acetic acid production. The amount of acetic acid produced is species/strain dependent, so some strains may benefit from more aeration without having the negative effect of creating too much acetic acid. Other strains may need a less aerobic starter (semi-aerobic) in order to produce the highest cell count with minimal acetic acid <ref>[http://www.ncbi.nlm.nih.gov/pubmed/12655458 Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. Aguilar Uscanga, Délia1, and Strehaiano. 2003.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0010(199712)75:4%3C489::AID-JSFA902%3E3.0.CO;2-9/abstract Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking. Maurizio Ciani and Luisa Ferraro. April 1999.]</ref><ref>[http://link.springer.com/article/10.1023%2FA%3A1014927129259 Acetic acid production by Dekkera/Brettanomyces yeasts. S.N. Feer. April 2002.]</ref>. In addition to acetic acid production, it has been observed that some ''Brettanomyces'' strains grown under aerobic conditions continue to produce THP when transferred to anaerobic conditions. See [[Tetrahydropyridine#Brettanomyces|THP]] for details.
This presents a sort of "catch 22" when growing ''Brettanomyces'' in a starter. The brewer must weigh the pros and cons of how much aeration to provide. If the ''Brettanomyces'' is going to be used in a [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]], for example, then a stir plate with foil covering the flask is the best choice. If the ''Brettanomyces'' is instead being pitched in secondary with the intention of long aging, then having a high cell count isn't as necessary and the risk of adding more acetic acid/ethyl acetate to an aging beer is greater. If a lot of acetic acid is produced during the starter, then they can opt to cold crash and decant the starter. ''Brettanomyces'' can have a difficult time flocculating and settling out, even when cold crashed. The brewer may need to allow a few days for the cells to fully sediment <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1099473923414154/?comment_id=1099522943409252&offset=0&total_comments=25&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Richard Preiss of Escarpment Yeast Labs on MTF. 6/26/2015.]</ref>. Additionally, ''Brettanomyces'' that is cold crashed may be slower to begin fermentation. If the brewer believes that the amount of acetic acid produced was insignificant, then cold crashing can be skipped and the entire starter can be pitched. Even if the starter has a lot of acetic acid, the amount of acetic acid in the volume of a starter is fairly insignificant once diluted into a full batch of wort or beer. If the starter is not going to be used within a month, then an aerobic starter is not the best option since the presence of a lot of acetic acid will slowly kill the ''Brettanomyces'' over time. In this case, the starter should be lightly shaken (or occasionally manually stirred), and an airlock put in place on the flask in order to keep out most of the oxygen.
Although more experiments are probably needed, agitation is believed to be an important factor for any species of microbe (yeast and bacteria). Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174865305875015&reply_comment_id=1176092372418975&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.]</ref>.
====Pitching Rate Calculators====
Current yeast pitching calculators for brewers are not adequate for determining ''Brettanomyces'' pitching rates based on starter volume size because the maximum cell density of ''Brettanomyces'' per mL of wort is 3 to 6 times the cell density of ''Saccharomyces''. For example, a given ''Saccharomyces'' strain may reach a cell density of 130 million cells per mL in a 1.040 wort (different ''Saccharomyces'' strains can have different cell densities as well, although they are a lot lower than ''Brettanomyces'' overall). Different ''Brettanomyces'' strain cell densities have been reported to be 600 to 885 million cells per mL in 1.040 wort depending on the species/strain <ref name="Yakobson_Propagation">[http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-results/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Results. Retrieved 2/17/2015]</ref><ref name="MarkTrent">[https://www.facebook.com/groups/MilkTheFunk/permalink/1114254011936145/ Conversation with Mark Trent and Lance Shaner on MTF regarding Brett pitching rates. 07-21-2015.]</ref>. Since yeast calculators are based on ''S. cerevisiae'' or ''S. pastorianus'' cell density, using one of these tools for ''Brettanomyces'' starters will create an unexpectedly high cell count in reality. There is not currently enough data to accurately determine starter volumes for ''Brettanomyces'', particularly because each strain and species have a different maximum cell density per mL of wort. However, pitching around 500-600 mL of a ''Brettanomyces'' starter for 5 gallons of 1.060 SG wort will achieve a pitching rate that is similar to lager yeast pitching rates, which has been recommended for [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]. [[Omega Yeast Labs]] is currently working on a project to create a more accurate ''Brettanomyces'' pitching rate calculator (it will also contain pitching rate calculations for specific strains of ''Saccharomyces'', which is something that current yeast pitching calculators do not include) <ref name="MarkTrent"></ref>.
Given this information, many brewers historically have been using the lager pitching rate settings in online yeast pitching calculators for ''Brettanomyces'' starters (around 2000 mL for 5 gallons, for example). Effectively, this means they have been pitching around 4 to 5 times the amount of ''Brettanomyces'' cells that they thought they were pitching. However, if this very high pitching rate is giving good results for brewers, it should continue to be used. Exploration of ''Brettanomyces'' pitching rates for 100% Brett fermentations is something to be desired once we know what our pitching rates actually are, and many brewers have been pitching 4-5 times the pitching rate for lagers if they use an online yeast pitching rate calculator instead of counting the cells under a [[Microscope|microscope]].
====[[Escarpment Laboratories]]====
* [https://escarpmentlabs.com/blogs/resources/how-to-choose-a-brett-strain-for-beer "How to Choose a Brett Strain For Beer."]
* Presentation by Richard Preiss:
: <youtube>NyGbnDMDn0Q</youtube>
===Pasteurization===
* [[Brettanomyces secondary fermentation experiment]]
* [[Brettanomyces Storage Survival Experiment]]
* [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]
* [[Crooked Stave Artisan Beer Project]]
* [[Scientific Publications]]