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updated all occurences of "Brett" to "Brettanomyces"
[[Image:Brettanomyces.jpg|thumb|200px|right|Brettanomyces]][[File:Brett-aroma-wheel.jpeg|thumb|200px|right|Brett Aroma Wheel by Dr. Linda Bisson and Lucy Joseph at UC Davis]]
'''Brettanomyces''', also known referred to by brewers as ''"Brett'' " or ''"Bretta''", is a yeast that was originally thought of as a spoilage yeast. The genus name ''Dekkera'' is used interchangeably with Brettanomyces, as it describes the teleomorph or spore forming form of the yeast <ref>[http://en.wikipedia.org/wiki/Brettanomyces Wikipedia. Brettanomyces. Retrieved 2/24/2015.]</ref>. Known for it's barnyard, fecal, horsey, metallic or Band-Aid flavors, ''BrettBrettanomyces'' was unwelcome in most breweries. However, in some styles like Saison and Lambic these flavors add a layer of complexity to the beer. ''BrettBrettanomyces'' can form a [[pellicle]]. See [[Lactobacillus]], [[Pediococcus]], [[Saccharomyces]], and [[Mixed Cultures]] charts for other commercially available cultures.
==Introduction of Characteristics==
''Brettanomyces'', first discovered by Hjelte Claussen in 1904, has traditionally been identified as a contaminate in wineries and brewers due to some of the phenols that it produces. These have generally been described as barnyard, burnt plastic, wet animal, fecal, and horse sweat <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 201.]</ref>. However, positive flavor components have been identified in beer such as pineapple, stone fruits, and to some degree acetic acid.
''BrettBrettanomyces'' has been identified on the skins of fruit <ref name="Schifferdecker"></ref>, as well as vectors (insects) <ref>[http://www.sciencedirect.com/science/article/pii/S0168160515001865 Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.]</ref>. ''BrettBrettanomyces'' is not considered to be airborne, however studies have found a very small amount of cells in the air at wineries where wine with ''BrettBrettanomyces'' in it was being handled (most of the yeasts found in the air were ''Aureobasidium'' and ''Cryptococcus'', which aren't considered spoilage organisms in beer and wine). These set of studies also determined that very specific methodology was needed in order capture Brett from the air, and indicated that the yeast was "stressed". <ref>[http://www.sciencedirect.com/science/article/pii/S0956713513002284 Screening of yeast mycoflora in winery air samples and their risk of wine contamination. E. Ocón, P. Garijo, S. Sanz, C. Olarte, R. López, P. Santamaría, A.R. Gutiérrez. Food Control Volume 34, Issue 2, December 2013, Pages 261–267.]</ref>. While it is possible for ''BrettBrettanomyces'' to be briefly carried by gusts of air, it only happens in the vicinity where the ''BrettBrettanomyces'' is actively fermenting. Good cleaning and sanitation, and cold temperatures should be employed to keep ''BrettBrettanomyces'' from infecting other equipment, and flying insects are a more likely cause for cross contamination of ''BrettBrettanomyces''.
It is common in scientific literature to see the names ''Dekkera'' and ''Brettanomyces'' used as the genus name, with ''Dekkera'' being the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph teleomorph] version and ''Brettanomyces'' being the [https://en.wikipedia.org/wiki/Teleomorph,_anamorph_and_holomorph anamorph]. There are five species within the genus of Brettanomyces: ''B. anomola'', ''B. bruxellensis'', ''B. custersianus'', ''B. nanus'', and ''B. naardenensis''. ''B. anomola'' and ''B. bruxellensis'' are the only two species that have been identified to have a teleomorph version; in their teleomorph version they are referred to as ''Dekkera anomola'' and ''Dekkera bruxellensis'' <ref name="Schifferdecker"></ref>. All of the other names such as the ones often used by yeast labs are derived by old nomenclature that is no longer used. The addition of the addition of small amounts of O2 stimulates glucose fermentation, as well as H+ acceptors such as acetaldehyde, acetone, pyruvic acid and other carbonyl compounds <ref name="yakobson_introduction">[http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad. The Brettanomyces Project. Introduction. Retrieved 8/11/2015.]</ref>.
Unlike most genres of yeast, ''BrettBrettanomyces'' has the characteristics of being very tolerant to high amounts of alcohol, 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 low nitrogen sources <ref name="Schifferdecker"></ref>. Perhaps the most differentiating characteristic of ''BrettBrettanomyces'' is its preference to ferment glucose in the presence of oxygen, which is the opposite preference in [[Saccharomyces]]. This was initially dubbed the "negative Pasteur effect" by Custers, and later the "Custers effect" <ref name="yakobson_introduction"></ref>.
Sulfite and SO<sub>2</sub> inhibits the growth of ''Brettanomyces'' <ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4549.2012.00702.x/abstract Removal of Brettanomyces Bruxellensis from Red Wine Using Membrane Filtration. Umiker, Descenzo, Lee, and Edwards. 04/24/2012.]</ref>.
| Ethyl valerate (Sweet, fruity, acidic, pineapple, apple, green, berry and tropical <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"> </ref> || 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"></ref>. 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>.
|-
| Isoamyl acetate (banana) || [[Acetic Acid]] and Isoamyl alcohol || 1.1ppm (flavor) <ref>[http://www.aroxa.com/beer/beer-flavour-standard/isoamyl-acetate/ Aroxa. Isaoamyl acetate. Retrieved 1/18/2015.]</ref> || C<sub>7</sub>H<sub>14</sub>O<sub>2</sub> <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/31276 PubChem. Isoamyl Acetate. Retrieved 08/25/2015.]</ref> || Produced by certain ''Saccharomyces'' strains but concentrations are generally reduced by ''Brettanomyces''. ''BrettBrettanomyces'' produces only very small amounts itself <ref name="Spaepen">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1982.tb04061.x/abstract Spaepen and Verachtert, 1982. Esterase Activity in the Genus ''Brettanomyces'']</ref>
|-
| Phenethyl acetate (sweet, honey, rose flower like) || Acetyl-CoA and 2-phenylethanol <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/7654 PubChem. Phenethyl Acetate. Retrieved 08/15/2015.]</ref><ref>[http://www.ymdb.ca/compounds/YMDB00574 YMDB. Phenethyl acetate.]</ref> || 3-5ppm (odor), 5-10ppm (flavor) <ref>[https://books.google.com/books?id=A8OyTzGGJhYC&pg=PA1521&lpg=PA1521&dq=Phenethyl+acetate+flavor+threshold&source=bl&ots=Ygvq_JFvhQ&sig=06Lj57fWYemcXizkEHnYzwfOXX8&hl=en&sa=X&ei=Zz-nVOPCKI61oQSFwIGABQ&ved=0CEAQ6AEwBA#v=onepage&q=Phenethyl%20acetate%20flavor%20threshold&f=false Burdock, George A]. Fenaroli's Handbook of Flavor Ingredients, Fifth Edition. CRC Press. 2005. pg 1521. </ref> || C<sub>10</sub>H<sub>12</sub>O<sub>2</sub> || Produced in very small amounts in Lambic <ref name="Spaepen"></ref><ref>[https://vtechworks.lib.vt.edu/bitstream/handle/10919/19203/Witrick_KA_D_2012.pdf?sequence=1 Characterization of aroma and flavor compounds present in lambic (gueuze) beer. Katherine A Thompson Witrick. 2012.]</ref>.
====Phenol Production====
Phenols such as 4-vinylphenol (4VP, barnyard, medicinal) and 4-vinylguaiacol (4-VG, clove) can be produced in beer by the decarboxylation of hydoxycinnamic acids, which are found in malt. While both ''Saccharomyces'' and ''Brettanomyces'' strains are have varying capabilities based on strain of converting hydroxycinnamic acids to their vinyl derivatives <ref>[http://www.mdpi.com/2304-8158/4/4/581/htm Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast. Michael Lentz and Chad Harris. 2015.]</ref>, ''Brettanomyces'' is also able to reduce these vinyl derivatives to ethyl derivatives. These vinyl derivatives have similar tastes to the ethyl derivatives but have lower flavor thresholds. Levels of all compounds produced vary depending on species and strain of ''BrettBrettanomyces''.
{| class="wikitable sortable"
| 4-Vinylphenol <ref name="Doss">[http://www.ahaconference.org/wp-content/uploads/presentations/2008/GregDoss_BrettBrewing.pdf Doss, Greg]. Brettanomyces:
Flavors and performance of single and multiple strain
fermentations with respect to time. Presentation at 2008 NHC. pg 12.</ref> <ref name="Yakobson_Michigan">[http://www.mbaa.com/districts/michigan/events/Documents/2011_01_14BrettanomycesBrewing.pdf Yakobson, Chad]. Brettanomyces in Brewing the horse the goat and the barnyard. 1/14/2011</ref> (Barnyard, 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 ''BrettBrettanomyces'' <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>.
|-
| 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> || 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> || C<sub>9</sub>H<sub>10</sub>O<sub>2</sub> <ref name="goodscents_4VG"></ref>. Produced by some strains of ''S. cerevisiae'' <ref>[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 ''BrettBrettanomyces'' 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>.
|-
| 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 ''BrettBrettanomyces'' <ref name="Oelofse"></ref>.
|-
| 4-Ethylphenol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Barnyard, Spicy, Smoky, Medicinal, Band-Aid <ref>[http://www.aroxa.com/wine/wine-flavour-standard/4-ethyl-phenol/ Aroxa Website. 4-Ethyl Phenol. Retrieved 08/19/2015.]</ref>) || Ethyl phenol || 4-vinylphenol || 0.3 ppm (odor; in beer) <ref name="Maarse">[https://books.google.com/books?id=_OvXjhLUz-oC&pg=PA513&lpg=PA513&dq=4-ethylphenol+odor+threshold&source=bl&ots=fzhA9yvvrJ&sig=K0QykyRqj9TnezG1Mih4gLru1ZE&hl=en&sa=X&ved=0CC4Q6AEwAmoVChMI77DfhNi1xwIV0jqICh1zVgQ8#v=onepage&q=4-ethylphenol%20odor%20threshold&f=false Volatile Compounds in Foods and Beverages. Henk Maarse. CRC Press, Mar 29, 1991. Pg 514, 515.]</ref> || C<sub>8</sub>H<sub>10</sub>O <ref name="pubchem_4EP">[http://pubchem.ncbi.nlm.nih.gov/compound/4-ethylphenol PubChem Website. 4-Ethylphenol. Retrieved 08/19/2015.]</ref> || Also known as 1-Ethyl-4-hydroxybenzene and P-Ethylphenol <ref name="pubchem_4EP"></ref>.
====Acid Production====
In the presence of oxygen, ''Brettanomyces'' strains are capable of producing acetic acid. 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 ''BrettBrettanomyces'' strains. Acetic acid produced by ''Brettanomyces'' may also be used in the synthesis of [[Secondary metabolites|acetate esters]] such as ethyl acetate. ''BrettBrettanomyces'' 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.
{| class="wikitable sortable"
====Two Approaches to Starters====
There are generally two approaches to handling ''BrettBrettanomyces'' 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://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 ''BrettBrettanomyces'' 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>.
This presents a sort of "catch 22" when growing ''BrettBrettanomyces'' in a starter. The brewer must weigh the pros and cons of how much aeration to provide. If the ''BrettBrettanomyces'' is going to be used in a [[100% Brettanomyces Fermentation]], for example, then a stir plate may be the best choice. If the ''BrettBrettanomyces'' 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, it is advised to cold crash and decant the starter. ''BrettBrettanomyces'' 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, ''BrettBrettanomyces'' 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.
Although more experiments and 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 Heit about starters and agitation. 11/09/2015.]</ref>.
====Pitching Rate Calculators====
Current yeast pitching calculators for brewers are not adequate for determining ''BrettBrettanomyces'' pitching rates based on starter volume size because the maximum cell density of ''BrettBrettanomyces'' per mL of wort is 3 to 6 times the cell density of ''Sacch''. For example, a given ''Sacch'' strain may reach a cell density of 130 million cells per mL in a 1.040 wort (different ''Sacch'' strains can have different cell densities as well, although they are a lot lower than ''BrettBrettanomyces'' overall). Different ''BrettBrettanomyces'' 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 off of ''Sacch'' cell density, using one of these tools for ''BrettBrettanomyces'' starters will create an unexpectedly high cell count in reality. There is not currently enough data to accurately determine starter volumes for ''BrettBrettanomyces'', particularly because each strain and species has a different maximum cell density per mL of wort. However, pitching around 500-600 mL of a ''BrettBrettanomyces'' 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 [[100% Brettanomyces Fermentation]]. [[Omega Yeast Labs]] is currently working on a project to create a more accurate ''BrettBrettanomyces'' pitching rate calculator (it will also contain pitching rate calculations for specific strains of ''Sacch'', 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 ''BrettBrettanomyces'' starters (around 2000 mL, for example). Effectively, this means they have been pitching around 4 to 5 times the amount of ''BrettBrettanomyces'' cells that they thought they were pitching. However, if this very high pitching rate is giving good results for brewers, it should continued to be used. Exploration of ''BrettBrettanomyces'' 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====
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://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://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 ''BrettBrettanomyces'' 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 ''BrettBrettanomyces'' 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>.
====Example of a Homemade Orbital Shaker====
===Storing Brett===
Long term storage of ''BrettBrettanomyces'' should be frozen with glycerol, rather than agar plates or slants, which have been observed anecdotally to reduce viability of ''BrettBrettanomyces'' over time <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1083075848387295/?comment_id=1083272091701004&offset=0&total_comments=13&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Matt Humbard, Ritchie Preiss, and Jeff Melo on MTF. 6/4/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1115817201779826&offset=0&total_comments=34&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Nick Impellitteri on MTF regarding storing Brett on agar plates. 7/24/2015.]</ref>. Chad Yakobson noted that after storing ''BrettBrettanomyces'' in a refrigerated environment (we don't know how Chad was storing the ''BrettBrettanomyces'' cultures when he observed this, for example on agar plates or slants or something else.), after 6 months the ''BrettBrettanomyces'' would die. If ''BrettBrettanomyces'' is stored cold, it will be very sluggish and slow to start fermentation. Non-pure cultures (such as beer bottle dregs with ''BrettBrettanomyces'' in it) should be stored refrigerated. Making a starter is highly recommended if the ''BrettBrettanomyces'' culture has been stored cold <ref>[http://youtu.be/AjVOzBtE27Y?t=43m Yakobson, Chad. Presentation at 2012 Music City Brew Off. At 43:00.]</ref>.
[[File:Brett viability temp storage.jpg|thumb|300px|[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/ Richard Preiss's results of storing Brett at different temperatures]]]
[[File:Brett storage MarkTrent.jpg|thumb|300px|[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1145139948847551&offset=0&total_comments=69&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Mark Trent repeated Richard Preiss's results of storing Brett at different temperatures, and also in different mediums.]]]
Yakobson's observations were not scientifically quantified and details of his process are lacking (how was the ''BrettBrettanomyces'' stored?), as far as we know. Richard Preiss of [http://www.escarpmentlabs.com/ Escarpment Yeast Labs] shared the results of a controlled experiment on MTF that showed that BSI's ''Brett brux Drie'' and ''B. claussenii'' survived better in low ABV beer when stored at refrigeration temperatures rather than room temperatures, contradicting the anecdotal observations reported by Yakobson. The samples were grown in 1.040 DME wort until typical cell density was reached, and measured for >95% viability after growth with trypan blue stain and microscopy. 10ml samples of each were stored in sterile conical tubes for one month at different temperatures (4°C and 24°C). The samples were burped to avoid having head pressure as a variable. Trypan blue stain and microscopy were used to measure the viability after one month. After one month of storage at 4°C (39.2°F), the viability of ''B. claussenii'' was 92%, and BSI Drie was 72% viability. The samples stored for one month at 24°C (75.2°F) showed a significant drop in viability, with ''B. claussenii'' ending up at 40% and BSI Drie at 6% viability. This experiment also indicates that the viability of ''BrettBrettanomyces'' strains/species after storage is strain/species dependent <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/ Richard Preiss Brett storage experiment results on MTF. 7/24/2015.]</ref>.
Mark Trent repeated Richard Priess's experiment, but tested different mediums (wort, liquid MYPG, and water). Two ''BrettBrettanomyces'' isolates, one from Orval and one from SARA Bernice, were grown in 10 degree Plato wort and MYPG. The isolates are labeled on the chart as "Brett O (Orval)" and "Brett T (Tim Clifford)" respectively. After growth was complete, 10 mL of aliquots were aseptically transferred to 15 mL centrifuge tubes. In addition, the Orval isolate was grown on a MYPG plate and 3 single colonies for each treatment were transferred to 1 mL of sterile RO water in a 2 mL glass tube. Each treatment was prepared in a duplicate and stored at either 22°C or 1°C. Viability was measured after 31 days. Data shown in the chart to the right. No other statistics were performed (there were no statistically significant differences between the different types of storage mediums at room temperature). All storage mediums shared results similar to Richard's results. This further shows that ''BrettBrettanomyces'' survival is a function of temperature, with lower temperatures being beneficial towards survival <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1145139948847551&offset=0&total_comments=69&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Mark Trent on MTF. 09/10/2015.]</ref>.
Questions raised by MTF members in regards to these results:
# How does warm storage versus cold storage affect agar plates and slants?
# Are there any unexpected results if the samples are stored longer (Richard will update), and would this change if the ''BrettBrettanomyces'' is periodically fed new sugars?
# How come using dregs from commercial beers stored at room temperature gives good results for brewers?