Changes

===Environment and Survival===

''Brettanomyces'' is commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries. These include structures such as wooden fermentation vessels, walls of the building, as well as the inside surface of wood barrels and actually buried within the wood of barrels. ''Brettanomyces'' has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and occasionally as deep as 5 to 8 mm, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and one study found that the ''Brettanomyces'' was able to penetrate the French oak barrels up to 8 mm, while only penetrate American oak barrels up to 4 mm) of the barrel <ref name="Agnolucci_2017" /><ref name="Cartwright_2018">[http://www.ajevonline.org/content/early/2018/05/23/ajev.2018.18024 Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam. Zachary M. Cartwright, Dean A. Glawe, Charles G. Edwards. 2018. DOI: 10.5344/ajev.2018.18024.]</ref>, with the highest concentration of surviving cells being at the top staves where oxygen is more accessible (although Cartwright et al. found the opposite was true, perhaps due to methodology of sampling or a difference in SO₂ concentrations). Some strains are able to utilize the cellulose of the wood as a carbon source, and occasionally form pseudohyphae within the wood which expands the surface area of the cells allowing them more access to nutrients and allowing them to survive in nutrient deficient environments <ref name="Cartwright_2018" />. Ozone gas has been shown to be an effective way to kill ''Brettanomyces'' that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of ''Brettanomyces'' on wine grapes. Liquid ozone has been shown to be less effective at eliminating ''Brettanomyces''. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing ''Brettanomyces'' within the wood of barrels (see [[Barrel#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment. Fabrizio, Vigentini, Parisi,Picozzi, Compagno, Foschino. 2015.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1466856417310068 Control of Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and gaseous ozone. Francesco Craveroa, Vasileios Englezos, Kalliopi Rantsiou, Fabrizio Torchio, Simone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. Although the role of ''Brettanomyces'' appears to be limited in distillation, it has been isolated during the fermentation process of tequila making. It has also been isolated from drains, pumps, transfer hoses, and other equipment that is difficult to sanitize. The survivability of ''Brettanomyces'' has also partly been attributed to its ability to form a [[Quality_Assurance#Biofilms|biofilm]] (in particular ''B. bruxellensis''). Microorganisms that can form a biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. ''Brettanomyces'' has therefore been identified as a significant contaminate for breweries and wineries. Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''B. bruxellensis''. ''Brettanomyces'' is also commonly found in sherry, and is found (although only rarely) in olive production, lemonade, kombucha, yogurt, pickles, and soft drinks. ''B. anomalus'' and ''B. bruxellensis'' are generally found much more commonly than the other three species of ''Brettanomyces'' <ref name="smith_divol_2016">[http://www.sciencedirect.com/science/article/pii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Brendan D. Smith, Benoit Divol. June 2016.]</ref>.

''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>.

A genetic survey of 145 different strains of ''B. bruxellensis'' from 29 countries, 5 continents, and 9 different fermentation niches was conducted in 2018 by Avramova et al. They found that these strains formed roughly 6 genetic groups with mostly separate ancestral lineages, and 1 group with a mixed ancestral lineage: 3 wine groups, 1 beer group, 1 kombucha group (most distantly related to the beer group), as well as 1 tequila/ethanol group that has multiple ancestral lineages <ref name="Avramova_2018" />. These groups are partially determined by the identification of at least two hybridization events that happened during the evolution of ''B. bruxellensis'', similar to the hybridization events that created the Saaz and Frohberg subgroups of ''S. pastorianus'' (the parents of these hybridization events in ''B. bruxellensis'', whether from different species or not, has yet to be determined and will require whole genome sequencing of species closely related to ''B. bruxellensis'') <ref name="Gounot_2019" />. This was expressed mostly in the ploidy level of each group (the number of sets of chromosomes), with 2 of the wine groups, the tequila group, and the beer group containing more sets of chromosome pairs than the other groups (diploid vs triploid; this is thought to encourage adaption and hybridization). Additionally, the triploid wine group was generally more tolerant of SO² than the diploid wine groups <ref name="Avramova_2018" />. A later study that also looked at the genome of older ''B. bruxellensis'' strains in bottles of wine going back as far as the year 1909 revealed that the SO² tolerant triploid strains only started appearing after the year 1990, which corresponds to when the wine industry started using SO² in most wine production (although it can also mean that the triploid strains are not as good at surviving in bottles of wine long-term compared to the diploid strains that have been isolated from much older bottles of wine; this will be determined to be the case or not in the future as bottles of wine from the 1990s continue to age). They also identified dozens of examples of wineries throughout France and Italy where the same strain of ''B. bruxellensis'' was found in multiple vintages of bottles of wine going back many decades, indicating that individual ''B. bruxellensis'' strains become long-term residents in wineries. Some identical strains have been found in different regions and even different continents, indicating that some strains have traveled not just due to traditional vectors such as insects or birds, but also probably due to human transportation such as wine bottle imports/exports or exchanges between industrial processes. This also indicates that while ''B. bruxellensis'' becomes rather sedentary and a constant resident in wineries, it can also adapt to the different winemaking conditions in different regions once it's been transported (different grapes, different climates, different fermentation temperatures, etc.), including adapting to improved modern hygienic practices such as higher SO² treatment. Overall, the results from this study suggest that ''Brettanomyces'' is able to adapt to living alongside certain human industries and has done so for at least a couple of centuries <ref name="Cibrario_2019">[https://www.biorxiv.org/content/10.1101/763441v1 Brettanomyces bruxellensis wine isolates show high geographical dispersal and long remanence in cellars. Alice Cibrario, Marta Avramova, Maria Dimopoulou, Maura Magani, Cécile Miot-Sertier, Albert Mas, Maria C. Portillo, Patricia Ballestra, View ORCID ProfileWarren Albertin, Isabelle Masneuf-Pomarede, Marguerite Dols-Lafargue. 2019. DOI: https://doi.org/10.1101/763441.]</ref>. The genetic differences between the fermentation substrates (beer, wine, etc.) were lower but still significant, and this was explained by the frequent cross-over of equipment such as wine barrels being used for beer fermentation. When comparing the geographic differences, they found geography contributed only 5% of genetic differences, while geography explained more than 50% of genetic differences in non-wine strains, suggesting that beer, kombucha, and tequila strains are more localized genetically than wine strains and that humans probably helped the wine strains travel across the globe. They also found that although one study reported spore-forming versions of ''B. bruxellensis'' (referred to as ''Dekkera bruxellensis''), the genetic makeup of the analyzed strains determined their ability to sporulate to be non-existent or rare (only one study that we know of by [https://link.springer.com/article/10.1007%2FBF02539015 Walt and Kerken in 1960] has reported sporulation in ''Brettanomyces'' only on specific agar types with vitamins added, indicating that sporulation in ''Brettanomyces'' is extremely rare) <ref name="Avramova_2018" />. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/2022801681081369/ Richard Preiss's discussion of this study on MTF].

==''Brettanomyces'' Metabolism==

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

===Carbohydrate Metabolism and Fermentation Temperature===

''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>.

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>.

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):

Glycosides are flavorless compounds often found in plants/fruits that are composed of a molecule (often a flavor active compound) bound to a sugar molecule. The glycosidic bond can be broken, releasing the sugar molecule and the potential flavor active compound. These bonds can be broken with exposure to acid, as well as specific enzymes (beta-glucosidase) which can be added synthetically or produced naturally by some microorganisms, including some strains of ''Brettanomyces'' that have beta-glucosidase enzyme activity (mostly ''B. anomalus'' strains) <ref>[https://en.wikipedia.org/wiki/Glycoside "Glycoside." Wikipedia. Retrieved 06/27/2016.]</ref>. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic <ref name="Daenen2">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00421.x/pdf Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. Luk Daenen, Femke Sterckx, Freddy R. Delvaux, Hubert Verachtert & Guy Derdelinckx. 2007.]</ref>. It is speculated that flavor compounds from hops can also be released from glycosides <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref>; however, at least one study has shown no significant difference in a blind taste test between hopped beer exposed to the beta-glucosidase enzymes and hopped beer that was not exposed to the enzyme <ref name="Vervoort">[http://onlinelibrary.wiley.com/wol1/doi/10.1111/jam.13200/abstract Characterization of the recombinant Brettanomyces anomalus β-glucosidase and its potential for bioflavoring. Yannick Vervoort, Beatriz Herrera-Malaver, Stijn Mertens, Victor Guadalupe Medina, Jorge Duitama, Lotte Michiels, Guy Derdelinckx, Karin Voordeckers, and Kevin J. Verstrepen. 2016.]</ref>.

See the [[Glycosides]] page for more details.

====Acid Production====

In the presence of oxygen, ''Brettanomyces'' species produce acetic acid as a byproduct of glucose fermentation. 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>. In an environment with oxygen present, 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). 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'' strains and is limited by limiting oxygen exposure (see [[Mixed_Fermentation#Aging|aging mixed fermentation beer]] for practical tips on limiting oxygen exposure). 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 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" />.

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>.