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==Brewing Techniques==
===Obtaining a clean culture and true attenuation ability===
Please note that some of the ''[[Brettanomyces]]'' cultures products sold are by certain labs have been reportedly contaminated with other microbes such as ''[[Saccharomyces]]'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1295691083792436 MTF discussion on 2016-04-30]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1309024112459133/ MTF Discussion, 2016-05-19]</ref><ref>[http://babblebelt.com/newboard/thread.html?tid=1108752780&th=1281111541 Chad Yakobson. The Burgundian Babble Belt Homebrew forum. 08/12/2010.]</ref>. Generally, ''Brettanomyces'' ferments slow and a fermentation could take considerably longer to ferment out compared to a fermentation containing ''Saccharomyces''(some specific strains of ''Brettanomyces'' are an exception to this and may fully ferment wort in the same or similar timeframe as brewers yeast). Besides Many strains of ''Brettanomyces'' has have a somewhat limited [[Brettanomyces#Brettanomyces_Metabolism|metabolism]] and the apparent attenuation would can be quite a bit lower compared to what a comparable ''Saccharomyces'' fermentation would showcase. Additionally, many strains of ''Brettanomyces'', especially many strains of ''B. anomalus'', cannot efficiently ferment maltose in brewing conditions, and therefore are not good candidates for 100% ''Brettanomyces'' beers (see [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|''Brettanomyces'' carbohydrate metabolism]] for details). If a ''Brettanomyces'' culture fully ferments out a beer in less than a month, then it may have a ''Saccharomyces'' contamination, however , there are exceptions to this (see [[100%25_Brettanomyces_Fermentation#Fermentation_Characteristics_of_Individual_Species_and_Strains|Fermentation Characteristics below]]). When using a yeast lab ''Brettanomyces'' product for 100% fermentation, it is advisable to contact the yeast lab regarding the ability of that strain(s) ability to fully ferment wort unless specifically noted on their product specification sheet/website.
===Starter Information===
===Fermentation Characteristics of Individual Species and Strains===
[https://link.springer.com/article/10.1007/s00217-022-04195-5 Cioch‑Skoneczny et al. (2023)] fermented a pale ale wort with and without grape must, pulp, and marc (pomice) using a single strain of ''B. bruxellensis'' (US-05 was used as a control). They found that the ''B. bruxellensis'' fermentations had a fermentation rate similar to US-05, although it took a bit longer to reach the same terminal gravity (while US-05 was finished fermenting after 9 days, the ''B. bruxellensis'' stalled after about day 6, and then started fermenting again at day 9 until reaching a similar final gravity around day 13). The ''B. bruxellensis'' was generally able to begin fermentation faster in the wine must/pulp/marc additions to wort. The ''B. bruxellensis'' consumed more FAN and was able to reduce malic acid, but it was less efficient at fermenting maltose than the US-05. This strain of ''B. bruxellensis'' did not express hyper-attenuation in these "100% Brett" fermentations <ref>[https://link.springer.com/article/10.1007/s00217-022-04195-5 Cioch-Skoneczny, M., Sral, A., Cempa, A. et al. Use of red grape pulp, marc and must in the production of beer. Eur Food Res Technol (2023). https://doi.org/10.1007/s00217-022-04195-5.]</ref>.
Not all species of ''Brettanomyces'' are effective at efficiently attenuating wort on their own. Additionally, some strains and species may produce better results flavor-wise than others.
Up until April 9, 2015, "WLP644 Brettanomyces bruxellensis Trois" was thought to be a ''Brettanomyces'' species. Following the analysis of the genetics of ''Trois'' by Lance Shaner and several other members of MTF that showed this strain to be ''S. cerevisiae'', White Labs released a statement saying that their DNA analysis also showed that Trois was a ''Saccharomcyes'' species, but they did not specify the species of ''Saccharomyces'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1100249870003226/ Archive of MTF discussions regarding Trois genetic analysis results.]</ref><ref>[http://www.whitelabs.com/blog/wlp644-research-results White Labs Blog article. April 9, 2015.]</ref>. Beer fermentations with the this strain (now labeled as "WLP644 - Saccharomyces Bruxellensis Trois") are no longer considered to be 100% ''Brettanomyces'' fermentations. While this strain does produce a lot of fruity esters, it does not produce phenols, which is a signifying characteristic of ''Brettanomyces'' fermentations. ''Trois'' fermentations are therefore not representative of the flavor profile of true ''Brettanomyces'' fermentations, and this has become a common misconception because of the popularity of ''Trois'' and the misclassification. See [https://www.facebook.com/groups/MilkTheFunk/permalink/1100249870003226/ this MTF thread] for links to the details about the efforts to identify WLP644 as ''S. cerevisiae'' from various independent sources.
WLP644 was sequenced in 2019 by the Hittinger Lab as part of a study into hybridisation of brewing yeasts and found to be pure ''S. cerevisiae'' - <ref>[https://beer.suregork.com/?p=4112]</ref> <ref>[https://www.nature.com/articles/s41559-019-0998-8]</ref><ref>[https://www.ncbi.nlm.nih.gov/sra/SRX6781781[accn]]</ref>.
When using WLP644, it is recommended to make a 1 liter starter for 36-48 hours due to the extremely small cell count of the vials <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1204614559566756/?comment_id=1204678676227011&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Lance Shaner on MTF. 12/30/2015.]</ref>.
There is surprisingly little data to back this idea up outside of the anecdotal information gathered from brewers fermenting with 100% Trois, which was once thought to be ''Brettanomyces'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1226859710675574/?comment_id=1226864927341719&reply_comment_id=1226949030666642&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Lance Shaner on MTF. 02/05/2016.]</ref>. One controlled experiment by [[Brettanomyces_secondary_fermentation_experiment|Lance Shaner of Omega Yeast Labs and Richard Preiss of Escarpment Labs]] showed that the levels of 4-ethyl guaiacol and 4-ethyl phenol produced by ''Brettanomyces'' did not depend on the amount of their 4-vinyl precursors, suggesting that ''Brettanomyces'' is capable of producing 4EP and 4EG ''de novo'' (without being dependent on precursors produced by ''Saccharomyces''). In addition to this, the possibility that brewers and even some yeast labs have ''Saccharomyces'' contamination issues in their ''Brettanomyces'' products complicates the issue. This is only one data point, however, and more data needs to be researched.
A study <ref name="Tyrawa_2017">[https://www.facebook.com/groups/MilkTheFunk/permalink/1285391951489016/ "Funky can be Great: Brettanomyces bruxellensis Beer Fermentations" (poster for study). Caroline Tyrawa, Richard Preiss, and George van der Merwe. 2017.] </ref> conducted by Caroline Tyrawa and Richard Preiss measured, among other things, the 4-ethyl guaiacol in 100% ''Brettanomyces bruxellensis''. It shows significant levels of 4-ethyl guaiacol in wort fermented by various strains of the before-mentioned yeast. Tyrawa also showed that there are higher levels of esters in 100% ''Brettanomyces'' fermentations compared to when ''Brettanomyces'' is co-fermented with ''S. cerevisiae'' (see [[Brettanomyces_and_Saccharomyces_Co-fermentation#Review_of_Scientific_Analysis_2|''Brettanomyces'' and ''Saccharomyces'' Co-fermentation]]. A somewhat speculative conclusion of this might be that the high ester levels of 100% ''Brettanomyces'' fermented beers might mask the "funky" flavor characteristics of phenols (4-ethylguaiacol, 4-ethylphenol, etc). As esters tend to be chemically unstable (ref?) the fruity character of a ''Brettanomyces'' beer will fade over time allowing the funk a more prominent role. This is also supported by a study that looked at 4-ethylphenol and 4-ethylguaiacol levels in one strain of ''B. bruxellensis'' when fermented alone and when co-fermented with a wine strain (EC1118); they found that there were about 20% more phenols in the 100% ''B. bruxellensis'' fermentation than there were when the ''B. bruxellensis'' was co-fermented with EC1118 (this might have been because the wine strain uesd, EC1118, can metabolize hydrocinnamic precursors differently and reduce the 4-vinyl levels <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1770814702946736/ Richard Preiss. Statements about the Kosel et al. study. Milk The Funk Facebook gruop. 07/26/2017.]</ref>) <ref>[https://academic.oup.com/femsyr/article-abstract/17/4/fox018/3867021/The-influence-of-Dekkera-bruxellensis-on-the?redirectedFrom=fulltext The influence of Dekkera bruxellensis on the transcriptome of Saccharomyces cerevisiae and on the aromatic profile of synthetic wine must. Janez Kose, Neža Čade, Dorit Schulle, Laura Carret, Ricardo Franco-Duarte Peter Raspor. 2017.]</ref>, however, Tyrawa's data looked at phenol levels over time and found that initially phenol levels were higher in 100% ''Brettanomyces'' fermentations compared to co-pitched with ''S. cerevisiae'' but over time the phenol levels in the co-pitch were slightly higher and phenol levels in general fluctuated quite a bit over the entire 21 day trial <ref name="Tyrawa_Masters">[https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation. University of Guelph, Masters Thesis. Department of Molecular and Cellular Biology. 2020.]</ref>. [https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert's Bachelors thesis] reported that after a 30-35 day primary versus secondary fermentation with different strains of ''B. bruxellensis'', the primary fermentations tended to produce a wider spread of phenols based on strain but overall produced less phenols than when the same strains were fermented in secondary after a primary fermentation with London Ale III <ref>[https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert for the degree of Honors Baccalaureate of Science in Chemical Engineering presented on May 21, 2021. Title: Performance of Brettanomyces Yeast Strains in Primary and Secondary Beer Fermentations.]</ref>.
Thomas Hübbe's masters thesis also supports the hypothesis that ''Brettanomyces'' produces more esters other than ethyl acetate when it is not co-fermented with ''S. cerevisiae'', specifically because it has better growth without competition from ''S. cerevisiae''. Although below threshold, the esters ethyl caprylate, ethyl caprate, ethyl dodecanoate, and ethyl tetradecanoate were significantly lower when ''Brettanomyces'' was co-fermented with ''S. cerevisiae'' and ''Lactobacillus'' than when it was fermented with only ''Lactobacillus''. Ethyl acetate (still under threshold levels) was higher when ''Brettanomyces'' was fermented with ''Lactobacillus'' but without ''S. cerevisiae'', and significantly higher when it was fermented with both ''Lactobacillus'' and ''S. cerevisiae'' <ref name="Hubbe" />. This seems to support the idea that, with the exception of the ester ethyl acetate, 100% Brettanomyces fermentations are not necessarily less phenolic, but that they are more fruity probably due to higher growth without competition from ''S. cerevisiae'' (although phenols were not measured in Hübbe's study) <ref>[https://www.facebook.com/groups/MilkTheFunk/1407620505932826/?comment_id=1407723619255848&comment_tracking=%7B%22tn%22%3A%22R8%22%7D Comments by Richard Preiss regarding Thomas Hübbe's masters thesis. 09/15/2016.]</ref>.