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updated all links to The Brettanomyces Project to use Wayback machine links
===Morphology===
The morphology of ''Brettanomyces'' can vary immensely from strain to strain (and species to species). Some strains can look similar in size and shape to ''S. cerevisiae'' under a microscopic image, while others are elongated or much smaller. This makes it difficult to identify ''Brettanomyces'' without DNA analysis (see [[Laboratory_Techniques#PCR.2FqPCR|PCR)]]. Morphologies of ''Brettanomyces'' grown on agar plates can also be different from strain to strain. For example, Devin Henry found that a sample of WLP648 that contained two closely related strains of ''B. bruxellensis'' grew completely differently on the same growth media. At first, larger, slightly off-white colonies grew on the plates (this was the first strain), and then a few days later the second strain grew as many smaller white-colored colonies. Other strains may appear as glossy or matted with jagged edges, etc. Morphology on agar plates can change depending on the type of growth media <ref>[http://web.archive.org/web/20240623073158/http://brettanomycesproject.com/dissertation/analysis-of-culturability-on-various-media-agar/morphological-traits/ Yakobson, Chad. "Morphological Trains". Masters Dissertation. 2011. Retrieved 05/12/2017.]</ref><ref name="bryan_vrai" /><ref>[https://eurekabrewing.wordpress.com/2012/03/27/brettanomyces-bruxellensis-microscopy-pictures/ Samuel Aeschlimann. "Brettanomyces bruxellensis microscopy pictures". Eureka Brewing blog. 03/12/2012. Retrieved 05/12/2017.]</ref>. While genetic (PCR) identification is required for any kind of confident identification of ''Brettanomyces'', specialized selective media can also help identify ''Brettanomyces''; see [[Laboratory_Techniques#Brettanomyces|Selective Media]].
See also:
* [http://web.archive.org/web/20240519145413/http://brettanomycesproject.com/2010/06/brettanomyces-yeast-cell-images/ ''Brettanomyces'' morphology examples from Remi Bonnart.]
* [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.]
===Carbohydrate Metabolism and Fermentation Temperature===
''Brettanomyces'' is able to ferment a wide range of sugars. All strains can ferment glucose, and many strains can ferment sucrose, fructose, and maltose, although at a slower rate than glucose. The ability of ''Brettanomyces'' to produce invertase enzyme which breaks sucrose down into glucose and fructose has been attributed to horizontal gene transfer from an unknown bacteria at some point in the evolution of ''Brettanomyces'' <ref name="roach_2019">[https://www.biorxiv.org/content/10.1101/805721v2 New genome assemblies reveal patterns of domestication and adaptation across Brettanomyces (Dekkera) species. Michael J. Roach, Anthony R. Borneman. 2019. DOI: https://doi.org/10.1101/805721.]</ref>. Some strains can also ferment galactose, mannose, ethanol, acetic acid, malic acid, and glycerol, although historically there are some contradicting studies in science regarding the specifics (more recent studies tend to use better methods), probably due to the genetic diversity of ''Brettanomyces'' species, and many previously published studies do not specify whether testing conditions were aerobic or anaerobic even though the availability of oxygen effects whether or not certain sugars can be fermented by a given strain of ''Brettanomyces'' <ref name="Steensels"></ref><ref name="smith_divol_2016"></ref><ref name="Smith_2018" />. For example, the species ''B. naardenensis'' can ferment a wide range of carbon sources, including galactose, maltose, xylose, trehalose, cellobiose, rhamnose, and arabinose <ref name="Tiukova_2019" /><ref>[https://www.cobbind.com.br/upload/trabalhos/t1arquivo/OMxtG7q2fxPzbRa1ZZuFnCnwNFh7.pdf INVESTIGATION OF THE POTENTIAL OF XYLOSE ASSIMILATION BY
BRETTANOMYCES BRUXELLENSIS. Jackeline M. Silva, Gilberto H. Teles, Ester Ribeiro & Will B. Pita. Department of Antibiotics, Federal University of Pernambuco, Recife, Pernambuco, Brazil. Aug 2024.]</ref>. Acetic acid, glycerol, succinic acid, and ethanol are only consumed if oxygen is present <ref name="smith_divol_2016"></ref>. The addition of H+ acceptors such as acetaldehyde, acetone, pyruvic acid, and other carbonyl compounds, stimulates anaerobic fermentation. Small amounts of oxygen also stimulate fermentation <ref name="yakobson_introduction">[http://web.archive.org/web/20240415090559/http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad. The Brettanomyces Project. Introduction. Retrieved 8/11/2015.]</ref>. The presence of small amounts of oxygen can allow some strains of ''Brettanomyces'' to utilize certain carbon sources. For example, several strains of ''B. bruxellensis'' can consume ethanol, glycerol, and acetic acid as food sources only when at least a low amount of oxygen is present (semi-aerobic conditions) and no other sugar is available. Acetic acid and glycerol are used as food sources by some strains only under fully aerobic conditions, but not under semi-aerobic or anaerobic conditions. It has been hypothesized that acetic acid and glycerol are only consumed by ''Brettanomyces'' when ethanol and other food sources are no longer available <ref name="Smith_2018" />.
''Brettanomyces'' strains may possess both alpha and beta-glucosidases. Beta-glucosidase is intracellular (works on sugars that are passed into the cell through the cell wall), while alpha-glucosidase is both intracellular and extracellular (released into the environment by the cell). <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref><ref name="Kumara_1993">[http://aem.asm.org/content/59/8/2352.short Localization and Characterization of α-Glucosidase Activity in Brettanomyces lambicus. H. M. C. Shantha Kumara, S. De Cort and H. Verachtert. 1993.]</ref> These enzymes allow ''Brettanomyces'' strains to break down a broad range of sugars, including long-chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to ''Saccharomyces'' yeasts. <ref name="Steensels"></ref><ref>[http://www.scribd.com/doc/277758178/Insight-into-the-Dekkera-anomala-YV396-genome Insight into the Dekkera anomala YV396 genome. Samuel Aeschlimann. Self-published on Eureka Brewing Blog. Spet 2015.]</ref>.
====Ester Production====
''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://web.archive.org/web/20240415090559/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" />.
| [[Ethyl acetate]] (fruity, pineapple, pear, solventy, nail polish remover) || [[Acetic Acid]] and ethanol || 33ppm (odor), 100ppm (flavor) || C<sub>4</sub>H<sub>8</sub>O<sub>2</sub> <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/ethyl_acetate PubChem. Ethyl Acetate. Retrieved 08/15/2015.]</ref> || High flavor threshold; pineapple or pear-like in low amounts and nail polish in high amounts. Increases production with higher temperatures and oxygen. Also produced by ''Saccharomyces'' species <ref name="Hubbe" />.
|-
| Ethyl butyrate (pineapple, mango, tropical fruit <ref>[http://www.flavoractiv.com/products/ethyl-butyrate-beer-flavour-standards/ Ethyl Butyrate Beer Flavour Standard. FlavorActIV. Retrieved 6/20/2015.]</ref>, juicy fruit gum <ref>Private corrospondance with Richard Preiss by Dan Pixley. 12/1/2016.</ref>) || [[Butyric Acid]] and ethanol || 0.4ppm (flavor) <ref>[http://www.flavoractiv.com/products/ethyl-butyrate-beer-flavour-standards/ Flavoractiv. Ethyl butyrate. Retrieved 1/18/2015.]</ref> || C<sub>6</sub>H<sub>12</sub>O<sub>2</sub> <ref name="pubchem_ethylbutyrate">[http://pubchem.ncbi.nlm.nih.gov/compound/ethyl_butyrate PubChem. Ethyl Butyrate. Retrieved 08/15/2015.]</ref> || Low levels of production by some species of Brettanomyces; production decreases with higher acidity <ref name="yakobson1">[http://web.archive.org/web/20240623080847/http://www.brettanomycesproject.com/dissertation/pure-culture-fermentation/pure-culture-fermentation-discussion/ Yakobson, Chad. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Pure Culture Fermentation Discussion. 2011.]</ref>. Also known as ethyl butanoate <ref name="pubchem_ethylbutyrate"></ref>.
|-
| Ethyl caproate (sweet, fruity, pineapple, banana, apple or aniseed) || Caproic acid and ethanol <ref>[https://books.google.com/books?id=1b1CAgAAQBAJ&pg=RA2-PA320&lpg=RA2-PA320&dq=Ethyl+caproate+precursors&source=bl&ots=myHXfoVz9f&sig=fHGkce4UmeJVC4M3Kk4TXUCO-Nc&hl=en&sa=X&ei=ip68VOqjFY-tyASpmoHoCA&ved=0CEQQ6AEwBA#v=onepage&q=Ethyl%20caproate%20precursors&f=false Encyclopedia of Food Microbiology. Batt, Carl A. Academic Press. Sep 28, 1999. Pg 320.]</ref> || 0.2ppm (flavor) <ref>[http://www.aroxa.com/beer/beer-flavour-standard/ethyl-hexanoate/ Aroxa. ethyl hexanoate. Retrieved 1/18/2015.]</ref> || C<sub>8</sub>H<sub>16</sub>O<sub>2</sub> <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/31265 PubChem. Ethyl Caproate. Retrieved 08/15/2015.]</ref> || Also known as Ethyl hexanoate, Ethyl butyl acetate, and butylacetate <ref>[http://www.chemspider.com/Chemical-Structure.29005.html Chemspider. Ethylhexanoat. Retrieved 1/18/2015.]</ref>. Can also be produced by ''Saccharomyces'' species <ref name="Hubbe" />.
| Ethyl isovalerate (fruity, sweet, berry-like with a ripe, pulpy fruit nuance, artificial grape <ref name="Fenaroli_ethylisovalerate">[https://books.google.com/books?id=15HMBQAAQBAJ&pg=PA638&lpg=PA638&dq=ethyl+valerate+threshold&source=bl&ots=avVr8PQQ_p&sig=zm81_lhLU86VJ4jBNnm4I9nnxDw&hl=en&sa=X&ved=0CDIQ6AEwBGoVChMImYrEl6usxwIVAjmICh1HGwEs#v=onepage&q=ethyl%20isovalerate%20threshold&f=false Fenaroli's Handbook of Flavor Ingredients, Fifth Edition. George A. Burdock. CRC Press, Dec 3, 2004. Pg 587.]</ref>) <ref name="Joseph"></ref><ref name="lucy_joseph"></ref><ref name="Lucy_2015" /> || [[Isovaleric Acid]] and ethanol || 30ppm (flavor) <ref name="Fenaroli_ethylisovalerate"></ref> || C<sub>7</sub>H<sub>14</sub>O<sub>2</sub> (same as ethyl valerate) <ref name="Fenaroli_ethylisovalerate"></ref> || Also found in pineapple, orange juice and peel oil, bilberry, blueberry, strawberry, Swiss cheese, other cheeses, cognac, rum, whiskey, sherry, grape wines, cocoa, passion fruit, mango, and mussels <ref name="Fenaroli_ethylisovalerate"></ref>. Also known as Ethyl 3-methylbutanoate <ref name="Joseph"></ref>. Not identified as a major product of ''B. bruxellensis'', but is produced in large quantities by some strains <ref name="Lucy_2015" />.
|-
| Ethyl lactate (fruity, creamy, rum <ref>[http://www.aroma-chemical.com/ethyl-lactate/ Best Aroma website. Ethyl Lactate. Retrieved 08/15/2015.]</ref><ref>[https://books.google.com/books?id=avYMy82EBuAC&pg=PA384&lpg=PA384&dq=ethyl+lactate+flavor&source=bl&ots=AZufxA6Htu&sig=rTbNo4rOSBY_6kuhGDtW_JqQ5oA&hl=en&sa=X&sqi=2&ved=0CD0Q6AEwBWoVChMI35jXjuirxwIVyKOICh0klgDF#v=onepage&q=ethyl%20lactate%20flavor&f=false Dictionary of Flavors. Dolf De Rovira. John Wiley & Sons, Feb 28, 2008. Pg 384.]</ref>) || [[Lactic Acid]] and ethanol || 0.2 ppm-1.66 ppm (odor) <ref>[http://hazmap.nlm.nih.gov/category-details?id=1179&table=copytblagents Haz-Map, Ethyl Lactate odor threshold.]</ref> || C<sub>5</sub>H<sub>10</sub>O<sub>3</sub> <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/7344 PubChem. Ethyl Lactate. Retrieved 08/15/2015.]</ref> || Increases production with higher amounts of Lactic Acid <ref>[http://web.archive.org/web/20240723012858/http://www.brettanomycesproject.com/dissertation/pure-culture-fermentation/impact-of-initial-concentration-of-lactic-acid/ Yakobson, Chad. The Brettanomyces Project. Impact of the Initial Concentration of Lactic Acid on Pure Culture Fermentation. Retrieved 6/16/2015.]</ref>
|-
| Ethyl valerate (Sweet, fruity, acidic, pineapple, apple, green, berry, tropical, bubblegum <ref name="Lucy_2015" /><ref name="goodscents_ethylvalerate">[http://www.thegoodscentscompany.com/data/rw1000701.html The Good Scents Company. Ethyl Valerate article. Retrieved 08/15/2015.]</ref>) <ref name="Joseph">[http://www.ajevonline.org/content/suppl/2015/07/28/66.3.379.DC1/Supplemental_Data.pdf Supplemental Data for: Joseph, C.M.L., E.A. Albino, S.E. Ebeler, and L.F. Bisson. Brettanomyces bruxellensis aroma-active compounds determined by SPME GC-MS olfactory analysis. 2015.]</ref><ref name="lucy_joseph">[http://slideplayer.com/slide/4473144/ Impact of Brettanomyces on Wine. Presentation by Lucy Joseph of UC Davis. Retrieved 08/15/2015.]</ref> || Valeric Acid (pentanoic acid) and ethanol || 1500-5000 ppm (odor) <ref name="Fenaroli_ethylvalerate">[https://books.google.com/books?id=15HMBQAAQBAJ&pg=PA638&lpg=PA638&dq=ethyl+valerate+threshold&source=bl&ots=avVr8PQQ_p&sig=zm81_lhLU86VJ4jBNnm4I9nnxDw&hl=en&sa=X&ved=0CDIQ6AEwBGoVChMImYrEl6usxwIVAjmICh1HGwEs#v=onepage&q=ethyl%20valerate%20threshold&f=false Fenaroli's Handbook of Flavor Ingredients, Fifth Edition. George A. Burdock. CRC Press, Dec 3, 2004. Pg 638.]</ref> || C<sub>7</sub>H<sub>14</sub>O<sub>2</sub> <ref name="goodscents_ethylvalerate" /> || Valeric acid quantities found in beer are minimal (0-1 ppm) and below odor threshold <ref>[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1974.tb03598.x/pdf Organoleptic Threshold Values of Some Organic Acids in Beer. Sigmund Engan. 1973.]</ref><ref>[https://books.google.com/books?id=allg4XxlOM4C&pg=PA91&lpg=PA91&dq=valeric+acid+beer&source=bl&ots=Pfb6EL9ufV&sig=sTb3gjpv7dlQNBOmGBPuDXJegLs&hl=en&sa=X&ved=0CB4Q6AEwAGoVChMIx9Kstp6sxwIVzCqICh2r7wc3#v=onepage&q=valeric%20acid%20beer&f=false Aroma of Beer, Wine and Distilled Alcoholic Beverages. L. Nykänen, H. Suomalainen. Springer Science & Business Media, May 31, 1983.]</ref>, and is probably also the case for Ethyl valerate. Ethyl valerate is also known as ethyl pentanoate <ref name="goodscents_ethylvalerate" />. Also found in apples, bananas, guava, stawberry, cheeses, rum, whiskey, cider, sherry, grape wines, cocoa, coffee, honey, and passion fruit <ref name="Fenaroli_ethylvalerate"></ref>. Not identified as a major product of ''B. bruxellensis'', but is produced in large quantities by some strains <ref name="Lucy_2015" />.
| Anomalus || ''Dekkera anomala'' || ''Brettanomyces anomalus'' || WLP640 || Typical barnyard funk with some fruitiness; claimed that it can be used for primary fermentation but a starter may be necessary. ||
|-
| Claussenii|| ''Dekkera anomala'' || ''Brettanomyces anomalus'' ||WLP645||Fruity, pineapple. Wine grape-like aroma, with light wood-like, floral, and citrus aromas. More fruit forward in the flavor, clean aftertaste with little to no "funk" <ref name="danpixley_mtf" />. || Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL <ref name="reddit_brett"></ref>. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1385144124847131/?match=YXR0ZW51YXRpb24sYXR0ZW51YXRlZCxjbGF1c3Nlbmlp this MTF thread] and [https://www.facebook.com/groups/MilkTheFunk/permalink/1309024112459133/?comment_id=1310955328932678&comment_tracking=%7B%22tn%22%3A%22R1%22%7D this MTF thread] which discuss the purity of this culture, and references [http://web.archive.org/web/20240623083404/http://brettanomycesproject.com/dissertation/pure-culture-fermentation/impact-of-pitching-rate/ Yakobson's data] that indicates that it does not attenuate wort efficiently when purely isolated.
|-
| Lambicus|| ''Dekkera bruxellensis'' || ''Brettanomyces bruxellensis'' ||WLP653||Horsey, Smoky, Spicy. High amount of ripe pineapple and overly ripe stone fruit in the aroma and flavor, with mild levels of blue cheese, leather, and spicy phenol in the flavor <ref name="danpixley_mtf" />. ||Different from WY's "lambicus". Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL <ref name="reddit_brett"></ref>.
====Two Approaches to Starters====
There are generally two approaches to handling ''Brettanomyces'' starters. The first is to use a stir plate set to a medium-high RPM with tin foil on top of the flask for 7-8 days, cold crash for a few days, and then decant the beer before pitching the sedimented yeast. The second approach is to use an orbital shaker set to 80 RPM to create a ''semi-aerobic'' environment (this means that the oxygen levels are low, but also not non-existent) for 7-8 days as described in ''The Brettanomyces project'' <ref name="chad_rpm">[http://web.archive.org/web/20240623090139/http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-methods/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Methods. Retrieved 2/18/2015.]</ref>, cold crashing can be skipped, and the entire starter is pitched into the wort. An alternative to the second approach is to use a stir plate on a very low setting so that only a very small "dimple" of a vortex is formed <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174867645874781&reply_comment_id=1174924805869065&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Mark Trent, Richard Preiss, and Roy Ventullo on MTF regarding creating a semi-aerobic starter without an orbital shaker. 11/06/2015]</ref>. If a stir plate is not available, give the starter an initial dosage of pure O2, and then cover it with foil so that oxygen can slowly diffuse into the starter, and gently agitate as often as possible <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1019859158042298/?comment_id=1020313737996840&offset=0&total_comments=24&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with Nick Impellitteri on MTF in regards to semi-aerobic starters. 2/16/2015.]</ref>.
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.
====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://web.archive.org/web/20240623065612/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 publicly available data that the authors of this wiki are aware of 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]].
====MYPG Growth Substrate and Other Laboratory Substrates====
For yeast laboratories, "Malt Yeast Peptone Glucose" growth substrate has been shown to be a better substrate than wort for initially growing ''Brettanomyces'' from a plate or slant. When grown in wort, ''Brettanomyces'' will often go through a 24 hour lag phase, a growth phase, another lag phase, and a second growth phase (all within 7-8 days). When grown in MYPG substrate, there is only a single growth phase and no lag phase, which has been reported by Yakobson to produce a larger cell count in the same amount of time <ref>[http://web.archive.org/web/20240519142207/http://www.brettanomycesproject.com/2009/08/mypg-vs-wort-as-the-growth-substrate/ Yakobson, Chad. The Brettanomyces Project. MYPG Compared to Wort as a Growth Substrate. Retrieved 2/18/2015.]</ref>. Cells grown in MYPG also are better adapted to grow in wort <ref>[http://web.archive.org/web/20240623065300/http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-discussion/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Discussion. Paragraph 5. Retrieved 2/18/2015.]</ref>. Practical instructions for making this substrate can be found on Jason Rodriguez's blog, "[http://sciencebrewer.com/2011/04/29/wild-yeast-project-mypg-culture-media/ Brew Science - Homebrew Blog]". Unfortunately, growing ''Brettanomyces'' pitches in MYPG for breweries isn't very practical due to needing almost 4 times the amount of MYPG versus wort to get the same pitching rate. In a brewery or homebrewery, using wort for ''Brettanomyces'' starters is more practical <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1150169708344575/ Conversation with Mark Trent, Lance Shaner, and Richard Preiss on MTF. 09/18/2015.]</ref>.
For other suggested substrates for growing ''Brettanomyces'' and potentially other yeasts, see [[Laboratory_Techniques#Brettanomyces|Laboratory Techniques]].
* [https://eurekabrewing.wordpress.com/2013/01/19/brettanomyces-genome-blasting/ Insight into the Brettanomyces Mitochondrial Genome, by Eureka Brewing Blog.]
* [https://catalogue.ncyc.co.uk/catalogsearch/result/?q=brettanomyces National Collection of Yeast Cultures in the UK - Database on what compounds different species/strains can ferment.]
* [http://web.archive.org/web/20240519133925/http://www.brettanomycesproject.com/ The Brettanomyces Project - Chad Yakobon's Brett research.]
* [http://www.themadfermentationist.com/p/commercial-cultures.html The Mad Fermentationist - Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions]
* [http://www.themadfermentationist.com/2014/11/phenols-and-brett-initial-results.html?m=1 The Mad Fermentationist - Comparison between English Ale yeast and Belgian Ale yeast primary fermentations, and Brett in secondary]