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''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" />.
fermentations with respect to time. Presentation at 2008 NHC. pg 12.</ref> <ref name="Yakobson_Michigan">[http://www.mbaa.com/districts/michigan/events/Documents/2011_01_14BrettanomycesBrewing.pdf Yakobson, Chad]. Brettanomyces in Brewing the horse the goat and the barnyard. 1/14/2011</ref> (Musty, Medicinal, Band-aid, Plastic) || Vinyl phenol || p-Coumaric Acid || 0.2 ppm (flavor; in beer) <ref>[http://www.scielo.br/scielo.php?pid=S1516-89132013000600018&script=sci_arttext Determination of 4-vinylgaiacol and 4-vinylphenol in top-fermented wheat beers by isocratic high performance liquid chromatography with ultraviolet detector. Mingguang Zhu; Yunqian Cui. Dec 2013.]</ref> || C<sub>8</sub>H<sub>8</sub>O <ref name="goodscents_4VP">[http://www.thegoodscentscompany.com/data/rw1005801.html The Good Scents Company. 4-Vinylphenol. Retrieved 08/18/2015.]</ref> || Production level is different across species/strains of ''Brettanomyces'' <ref name="Oelofse">[http://www.sciencedirect.com/science/article/pii/S0740002008002050 Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production. A. Oelofse, A. Lonvaud-Funel, M. du Toit. 2009.]</ref>. Coumaric acid levels vary greatly between barley varieties; for example, between 320 µg/kg to 950 µg/kg in different varities of barley husks and 73 µg/kg to 657 µg/kg in different varities of barley malt <ref name="Cortese_2020" />. Coumaric levels are generally higher in barley malt than they are in wheat malt. Coumaric acid is stable through the wort boiling process <ref name="kalb_2021" />.
It's also been demonstrated that the presence of p-coumaric can assist in reviving so-called [[Quality_Assurance#Viable_But_Nonculturable|VNBC cells of ''B. bruxellensis'']], suggesting that ''Brettanomyces'' can use energy sources such as p-coumaric acid to maintain survival in nutrient poor conditions <ref>[https://www.mdpi.com/2306-5710/9/3/69 Chandra M, Branco P, Prista C, Malfeito-Ferreira M. Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis. Beverages. 2023; 9(3):69. https://doi.org/10.3390/beverages9030069.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/posts/7289633364398148/?comment_id=7295436620484489 Richard Preiss. Milk The Funk Facebook group thread about p-coumaric acid metabolism. 08/27/2023.]</ref><ref>[https://www.mdpi.com/2306-5710/9/3/69 Chandra M, Branco P, Prista C, Malfeito-Ferreira M. Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis. Beverages. 2023; 9(3):69. https://doi.org/10.3390/beverages9030069.]</ref>.
|-
| 4-Vinylguaiacol <ref name="Doss"></ref><ref name="Yakobson_Michigan"></ref> (Clove) || Vinyl phenol || Ferulic Acid || 0.3 ppm (flavor; in beer) <ref>[http://www.aroxa.com/beer/beer-flavour-standard/4-vinyl-guaiacol/ Aroxa Website. 4-Vinylguaiacol. Retrieved 08/19/2015.]</ref> || C<sub>9</sub>H<sub>10</sub>O<sub>2</sub>. Also known as 2-methoxy-4-vinyl phenol <ref name="goodscents_4VG">[http://www.thegoodscentscompany.com/data/rw1005101.html The Good Scents Company. 2-methoxy-4-vinyl phenol. Retrieved 08/18/2015.]</ref>. || Produced by some strains of ''S. cerevisiae'' (see [[Saccharomyces#Phenolic_Off_Flavor_Strains|''Saccharomyces'']]) <ref name="Coghe_2014">[http://pubs.acs.org/doi/abs/10.1021/jf0346556 Ferulic Acid Release and 4-Vinylguaiacol Formation during Brewing and Fermentation: Indications for Feruloyl Esterase Activity in Saccharomyces cerevisiae. Stefan Coghe, Koen Benoot, Filip Delvaux, Bart Vanderhaegen, and Freddy R. Delvaux. 2004.]</ref>. Some ''Brettanomyces'' species/strains may also be able to produce this compound at varying levels <ref name="Joseph"></ref><ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1995.tb07374.x/abstract The biotransformation of simple phenolic compounds by Brettanomyces anomalus. Duncan A.N. Edlin1, Arjan Narbad, J. Richard Dickinson1 andDavid Lloyd. 2006.]</ref><ref name="Oelofse"></ref>. Organic malts have been linked to higher levels of 4VG, vanillan, and their malt precursor ferulic acid <ref name="Iyuke_2008">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.2008.tb00773.x/full The Effect of Hydroxycinnamic Acids and Volatile Phenols on Beer Quality. S. E. Iyuke, E. M. Madigoe, and R. Maponya. 2008.]</ref>. Ferulic acid is released during mashing, with an optimal mash temperature of 40-45°C (104-113°F) and a mash pH of 5.7-5.8 (enyzmatic release of ferulic acid is optimal at a pH of 7.5, but this high of a pH is difficult to achieve during mashing and would cause other enzymatic problems during the later steps of the mash) <ref name="Coghe_2014" /><ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/j.2050-0416.2000.tb00036.x Extraction and Assay of Ferulic Acid Esterase From Malted Barley. F. J. Humberstone D. E. Briggs. 2012.]</ref>. 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>.
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]].
* [[Brettanomyces secondary fermentation experiment]]
* [[Brettanomyces Storage Survival Experiment]]
* [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]
* [[Crooked Stave Artisan Beer Project]]
* [[Scientific Publications]]