View source for Brettanomyces Fermentation

'''100% ''Brettanomyces'' fermentations''' are beers that are fermented with only ''Brettanomyces'' and no other microbes such as ''S. cerevisiae'', ''Lactobacillus'', or ''Pediococcus''. 

==General Information==
The method of fermenting wort with only ''Brettanomyces'' was pioneered by Tomme Arthur from Pizza Port/Lost Abbey, and Peter Bouckaert from New Belgium in 2004 with their 100% ''Brettanomyces'' fermentented ''Mo' Bretta'', and Vinnie Cilurzo of Russian River with Sanctification later that same year <ref>[http://www.amazon.com/American-Sour-Beer-Innovative-Fermentations-ebook/dp/B00LV8IRRI/ref=dp_kinw_strp_1 American Sour Beers.  Michael Tonsmeire.  July 2014.  Pg 189.]</ref>.  Avery Brewing Co. and Jeff O'Neal from Ithica Beer Co. also produced early 100% ''Brettanomyces'' beers <ref>[https://youtu.be/IGzoh4brILA?t=20m59s Yakobson, Chad.  Interview on Craft Commander.  12/20/2016.  Retrieved 12/20/2016.] (~21 mins in)</ref>.  This method was further popularized by Chad Yakobson's ''Brettanomyces'' Dissertation on the [http://www.brettanomycesproject.com/dissertation/ Brettanomyces Project blog], and by his brewery, [[Crooked Stave Artisan Beer Project]].  While primary fermentation with ''Brettanomyces'' is a complex subject due to the wide range of characteristics of different species and strains of ''Brettanomyces'', it is believed that beer that is fermented with ''Brettanomyces'' in primary usually produces a surprisingly clean, lightly fruity beer (see Chapter 8 in ''American Sour Beers'' by Michael Tonsmeire for a full description of 100% ''Brettanomyces'' fermented beers).  However, much of this belief was partly based on the misclassification of what is now referred to as "''Saccharomyces'' Trois".  The issue of characterizing 100% ''Brettanomyces'' fermentations is further complicated by the fact that some sources of ''Brettanomyces'' also contain ''Saccharomyces'' or other unadvertised microbes.  Furthermore, most ''Brettanomyces'' strains cannot fully ferment wort due to not being able to utilize maltose (see the [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|''Brettanomyces'']] wiki page and [http://brettanomycesproject.com/dissertation/pure-culture-fermentation/impact-of-pitching-rate/ Chad Yakobson's Dissertation]).  

Typical characteristics of ''Brettanomyces'' primary fermentations (these are generalizations, and may not be true for every strain):
* Most pure cultures of ''Brettanomyces'' cannot fully attenuate wort due to not fermenting maltose, especially under anaerobic conditions.
* Initially subdued  "horsey", "funky", "barnyardy" flavors due to the lack of ''Saccharomyces'' esters/phenols (see the [[Brettanomyces#Brettanomyces_Metabolism|Brettanomyces Metabolism]] page for more information).  However, this is a generalization and some brewers have reported getting some "funkier" flavors out of some strains.  
* Light fruit characteristics.
* A longer lasting hop aroma and flavor due to Brett's ability to constantly metabolize micro-oxygenation. 
* A lack of glycerol, which is a compound that ''Saccharomyces'' produces which gives beer it's slick mouthfeel.  Malts such as oats or flaked wheat are often used to make up for the lack of glycerol.  However, the role of glycerol in creating mouthfeel is debatable in the wine world <ref>[https://www.winesandvines.com/features/article/68760 Tim Patterson.  "Many Roads to Mouthfeel".  Wines & Vines Magazine.  Nov 2009.  Retrieved 03/23/2018.]</ref>.
* Slightly longer primary fermentation in general (3-6 weeks), although some people have reported faster fermentations between 1-3 weeks for some strains and conditions (lower starting gravity beers, for example) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1158650034163209/ Conversation on MTF regarding how long 100% ''Brettanomyces'' ferments can take.  10/04/2015.]</ref>.  
* Perceived bitterness may be quite a bit lower than the same wort fermented with a clean ale yeast.

==Brewing Techniques==
===Obtaining a clean culture and true attenuation ability===
Please note that some of the ''[[Brettanomyces]]'' products sold 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). Many strains of ''Brettanomyces'' have a somewhat limited [[Brettanomyces#Brettanomyces_Metabolism|metabolism]] and the apparent attenuation 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===
When relying on a ''[[Brettanomyces]]'' culture for primary fermentation, a starter will often be necessary due to the fact that most yeast labs provide a small cell count for their ''[[Brettanomyces]]'' cultures.  See the [[Brettanomyces#Starter_Information|Brettanomyces Starter Information]] section for more information on ''Brettanomyces'' starters. About 500ml starter per 25 liters of wort seems to be the current best practice.  Data from Thomas Hübbe supports that the initial pitching rate doesn't have a great effect on the final cell count in pure ''Brettanomyces'' starters or beer, indicating that ''Brettanomyces'' is fairly forgiving in regards to small initial cell counts <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/files/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis).  Thomas Hübbe.  2016.]</ref>.

See also [[Brettanomyces#Pitching_Rate_Calculators|''Brettanomyces'' pitching rates]].

===Wort Production===
American IPA or American Pale Ale recipes are a tried and true general approach to making wort that is favorable to 100% ''Brettanomyces'' fermentations.  Fruitier hops such as citra, amarillo, galaxy, etc. tend to compliment the light fruity characteristics of a ''Brettanomyces'' primary fermentation.  The addition of body-increasing malts such as oats, unmalted barley, rye, wheat, or carapils may assist with the lack of glycerol that is typical for ''Brettanomyces'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1177290552299157/?comment_id=1177304778964401&reply_comment_id=1177435872284625&total_comments=4&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Tom Belgrano on MTF.  11/12/2015.]</ref>, but isn't always necessary.  Otherwise, wort production can remain the same as it is for an American IPA/Pale Ale recipe.  Aeration of the wort before fermentation starts should be done.  This will greatly increase cell growth (see the [[Brettanomyces Propagation Experiment]]).  As far as we know, acetic acid is a byproduct of ethanol production by ''Brettanomyces'' and not the prior lag phase, so as long as ethanol is not already being produced then acetic acid production is not a concern <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1204641279564084/?comment_id=1204674032894142&reply_comment_id=1204765566218322&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Richard Preiss on MTF about oxygenating wort that will receive 100% Brett.  12/30/2015].</ref>.  Examples of commercial 100% ''Brettanomyces'' beers that receive the same amount of initial aeration that would be typical of ales of their respective gravities are "Sanctification" from Russian River and "Mo’ Betta Bretta" from Lost Abbey <ref>[https://byo.com/hops/item/262-brettanomyces BYO Magazine.  Brettanomyces.  Steve Piatz.  October 2005.]</ref>.

===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.

* Some microbiologists have witnessed that ''B. claussenii'' is very slow to ferment wort by itself.  If fermentation finishes in two weeks, this might be due to contamination of another yeast <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1278391665522378/?comment_id=1278392922188919&reply_comment_id=1279087198786158&comment_tracking=%7B%22tn%22%3A%22R%22%7D  Conversation with Lance Shaner on MTF regarding ''B. claussenii'' attentuation.  04/06/2016.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1385144124847131/?comment_id=1385326081495602&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Brian Martyniak regarding general ''Brettanomyces'' sugar utilization.  08/24/2016.]</ref>.

* Not all strains can ferment maltose, which is almost 50% of the sugar composition of wort.  These strains should be avoided for 100% ''Brettanomyes'' fermentations.  See [[Brettanomyces#Carbohydrate_Metabolism|''Brettanomyces'' carbohydrate metabolism]] for more details.

* [http://brettanomycesproject.com/dissertation/pure-culture-fermentation/impact-of-pitching-rate/ Chad Yakobson's thesis] showed that WLP645, WLP650, WLP653, WY5112, WY5526, and WY5151 were not able to attenuate wort more than 50% within 35 days (these were pure cultures).  BSI Drie was the only strain tested that was able to attenuate wort at levels similar to brewers yeast.  All strains that he tested were able to utilize maltose, however some less efficiently than others.  More time may or may not have resulted in further attenuation.  Contamination with another yeast is one explanation for why brewers are able ot use these cultures from labs to fully attenuate wort (Yakobson used purified isolates for this research).

* [https://www.facebook.com/groups/MilkTheFunk/permalink/1935201276508077/ Nick Mader of Fremont Brewing (2017 Master Brewers Conference Presentation)] observed that 100% BSI Drei fermentation resulted in around 77% attenuation (3.17°P final gravity), while co-fermentation with different pitch rates of a saison yeast resulted in around ~90% attenuation (~1.5°P final gravity).  The esters were generally lower than when cofermented with the saison yeast, but ethyl decanoate (apple, brandy) was considerably higher with the 100% fermentation with BSI Drei.  4-ethyl phenol concentrations with 100% Drei were around the same as when cofermented with the saison yeast. See also [[Brettanomyces_and_Saccharomyces_Co-fermentation#Review_of_Scientific_Analysis|cofermentation with ''Saccharomyces'']].

* [[Brettanomyces_Propagation_Experiment|Mark Trent's ''Brettanomyces'' propagation experiment]] tested his house strain of ''Brettanomyces'' (originally isolated from Orval), which fully attenuated wort under different different conditions within 6 days.  So, there are strains that are faster fermenters, but they appear to be the exception to the rule.  

* [https://www.facebook.com/groups/MilkTheFunk/permalink/1285391951489016/ Effects of temperature on final gravity with 100% ''Brettanomyces'' by strain (data provided by Escarpment Labs).]

* [https://www.facebook.com/groups/MilkTheFunk/permalink/1222754581086087/ Anecdotal finishing gravities of different strains reported on MTF] and  [https://www.facebook.com/groups/MilkTheFunk/permalink/1309024112459133/?comment_id=1309105312451013&comment_tracking=%7B%22tn%22%3A%22R4%22%7D comments on the reliability of anecdotal data by Lance Shaner].

* See attenation rates based on pitch rate from [http://www.whitelabs.com/sites/default/files/R%26D%20Wild%20Yeast%20and%20Bacteria%20Experiments_2.pdf this White Labs data sheet].

* In general a broad selection of various ''Brettanomyces'' yeasts and a few months of time is a safe bet to make sure fermentation carries through.

* [https://www.facebook.com/groups/MilkTheFunk/permalink/2062107787150758/ MTF threads on anecdotal experiences of different strains.]

===Impact of Fermentation Temperature===
[[File:Tyrawa attenuation 2019.JPG|thumb|360px|[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.565 '''A)''' Fermentation at 15°C (59°F) and '''B)''' at 22.5°C (72.5°F) by different strains of ''B. bruxellensis'' and Fermentis US-05; the "PEST" strains are wine strains while the others are beer strains.  Source: The temperature dependent functionality of ''Brettanomyces bruxellensis'' strains in wort fermentations, by Tyrawa et al (2019).]  <p>Click [https://www.facebook.com/groups/MilkTheFunk/permalink/1285391951489016/ here] for more charts and graphs from this study.</p>]]

In ''Saccharomyces'' species, higher fermentation temperature has been associated with faster fermentation, higher growth rates, and ester formation.  [https://onlinelibrary.wiley.com/doi/full/10.1002/jib.565 Tyrawa et al. (2019)] set out to explore the impact of fermentation temperature on 7 beer strains (including BSI Drei and several isolates from commercial sour/saison/lambic beers) and 2 wine strains of ''B. bruxellensis''.  Fermentis US-05 and the BSI Drei were used as controls.  Each strain was fermented in autoclaved 100% 2-row malt wort at a starting gravity of 1.050 and fermented at 15°C (59°F) versus 22.5°C (72.5°F) for 28 days.  The pitching rate was 1.2 x 10<sup>7</sup> cells/mL.  Each strain was genetically tested to ensure their species was correctly identified and that they were genetically distinct from each other <ref name="Tyrawa_2019">[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.565 The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations.  Caroline Tyrawa, Richard Preiss, Meagan Armstrong, George van der Merwe.  2019.  DOI: https://doi.org/10.1002/jib.565.]</ref>.  The fermentation temperature of 30°C (86°F) was also briefly examined, but they were described as "smelling terrible" by Richard Preiss, and so were discarded from the 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>.

Their results showed that there is a vast diversity in how temperature effects attenuation for different strains of ''B. bruxellensis''.  In general, the cooler 15°C (59°F) fermentation temperature slowed the attenuation rate for most strains.  The US-05 attenuated the most at both temperatures, with only one saison strain matching that attenuation level when fermented at 22.5°C (72.5°F).  This same strain, which was isolated from a commercial USA saison beer, and the BSI Drei strains had fast attenuation rates that were comparative to the US-05 fermentation at both temperatures, while the other strains had lag times of 8-10 days at 15°C (59°F) or 2-4 days at 22.5°C (72.5°F).  Additionally, the colder temperature resulted in a wide variance between strains and their ability to ferment different types of sugars.  Glucose and fructose were the only sugars fermented by all strains at the lower fermentation temperature by all of the strains, with a lot of variation for fructose, sucrose, maltose, maltotriose, cellobiose, and maltodextrin.  Only BSI Drei and both of the wine strains were able to ferment cellobiose at the colder fermentation temperature (several of the saison strains began fermenting cellobiose at the warmer temperature, while others did not), indicating that colder temperatures can greatly limit or even eliminate the ability to ferment cellobiose in most strains, and maybe the environment from which the strains were isolated from determines the efficiency to ferment different types of sugars for different strains of ''B. bruxellensis'' <ref name="Tyrawa_2019" />.  

At 15°C (59°F), none of the ''Brettanomyces'' strains could match the US-05 attenuation, with most of them falling to around 25-50% less final attenuation after 28 days, and one of the wine strains and one of the USA saison strains not fermenting at all.  Still, this data showed that some beer strains of ''B. bruxellensis'' can ferment at lower temperatures.  Interestingly, one of the wine strains was almost unaffected by the difference in fermentation temperature; it only lagged for a couple of days longer in the colder 15°C (59°F) fermentation temperature versus the warmer 22.5°C (72.5°F) fermentation temperature, but achieved the same amount of attenuation after 28 days <ref name="Tyrawa_2019" />.

At 22.5°C (72.5°F), all of the ''Brettanomyces'' strains fermented more efficiently, although their final attenuation numbers for some strains were significantly less than other strains, with only one strain (the previously mentioned strain that was isolated from a commercial USA saison beer) attenuating at levels that matched the US-05 control.  Three strains (one wine strain and two beer strains) attenuated just over half of the rate as the more successful fermenters.  This indicates that most ''B. bruxellensis'' strains are not as efficient at fermenting wort by themselves as ''Saccharomyces cerevisiae'' ale strains, and there is a lot of diversity between ''B. bruxellensis'' strains on how efficiently they can ferment wort <ref name="Tyrawa_2019" />.

The effect on phenol production, 4-ethylguaiacol (clove) and 4-ethylphenol (barnyard), was relatively the same and above flavor threshold for both fermentation temperatures for all of the ''B. bruxellensis'' strains tested, although some strains had slightly more or less of these phenols produced at the different fermentation temperatures.  By comparison, the temperature of the fermentation had a much larger impact on the amount of esters produced.  Ethyl acetate (pineapple/pear) was significantly higher in the warmer fermentation temperature of 22.5°C (72.5°F) than the cooler temperature of 15°C (59°F) for all strains, with one saison strain producing significantly more ethyl acetate and another saison strain producing significantly less ethyl acetate than the other strains.  As expected, the US-05 produced higher amounts of isoamyle acetate (banana) at 22.5°C (72.5°F) and lower amounts at 15°C (59°F).  The US-05 produced comparably high amounts of phenethyl alcohol (dried rose), phenethyl acetate (honey/rose pedal), and isoamyl alcohol (banana/oily) at both temperatures. These esters were generally not produced at more than very low levels by the ''Brettanomyces'' strains.  The phenol 4-vinylguaiacol was produced more at the lower temperature by BSI Drei and one of the saison strains, indicating that the lower fermentation temperature slowed the process of these strains to convert the 4-VG to 4-EG.  They also produced the lowest amount of the ethyl phenols compared to the other ''Brettanomyces'' strains.  All of the ''Brettanomyces'' strains isolated from beer produced other fatty acid esters at significant levels above tastes threshold that the US-05 produced below tastes threshold.  These esters included ethyl caproate (pineapple/apple), ethyl caprylate (pineapple), ethyl decanoate (brandy/apple) and ethyl nonanoate (fruity/rose/waxy).  In general, a higher amount of these esters were produced at the higher fermentation temperature, although there were exceptions.  Several of the saison strains and the lambic strain produced higher amounts of esters than the BSI Drei control, especially when fermented at the warmer temperature, demonstrating the amount of esters produced is highly variable among different strains of ''B. bruxellensis'', particularly when fermented at 22.5°C (72.5°F) rather than the lower fermentation temperature of 15°C (59°F).  Interestingly, the two wine strains of ''Brettanomyces bruxellensis'' did not produce above threshold levels of any of these esters at either fermentation temperature (the wine strains did produce the highest levels of decanoic acid, which was elevated at the higher fermentation temperature versus the lower fermentation temperature) <ref name="Tyrawa_2019" />.

See also:
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1285391951489016/ Richard Preiss summarizes his study and provides the original poster with charts and graphs of the data on Milk The Funk.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2710564272305103/ More Q&A with Richard Preiss on Milk The Funk regarding the full published paper.]
* [[100%25_Brettanomyces_Fermentation#Are_100.25_Brett_Beers_Really_Cleaner.3F|Are 100% ''Brettanomyces'' Beers Really Cleaner?]]

===Aging===
In general, 100% ''Brettanomyces'' beers are not aged for more than 2-3 months (sometimes less).  The beer can be packaged when it reaches a stable final gravity (see [[Packaging#Final_Gravity|Packaging]]).  "Brett IPA's", for example, are often not aged since this leads to a decline in hop flavor and aroma.  However, there is no "rule" against aging them if the brewer chooses to.  Due to the potential for acetic acid development when exposed to oxygen over time, care should be taken when aging any beer with living ''Brettanomyces''.  See [[Mixed_Fermentation#Aging|mixed fermentation aging]].

===Styles===
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2033652283329642/ MTF thread on Brett NEIPA (also includes techniques for IPA's that are brewed with ''S. cerevisiae'' first, and then ''Brettanomyces'' for a second fermentation).]

==Questioning Conventional Wisdom==

===About Trois===
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>.

===Are 100% ''Brettanomyces'' Beers Really Cleaner?===
A lot of the conventional wisdom listed above regarding 100% ''Brettanomyces'' fermentations is anecdotal information derived from Trois fermentations.  As explained above, Trois is not actually ''Brettanomyces'', and so conventional wisdom regarding 100% ''Brettanomyces'' beers has been brought into question.  One particular area of question is the conventional wisdom that ''Brettanomyces'' requires phenols from POF+ ''Saccharomyces'' strains in order to convert 4-vinyl phenols into 4-ethyl phenols, and that 100% ''Brettanomyces'' fermentations are therefore "less funky".  

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>.

==See Also==
===Additional Articles on MTF Wiki===
* ''[[Brettanomyces]]''
* [[Brettanomyces and Saccharomyces Co-fermentation]]
* [[Brettanomyces Propagation Experiment]]
* [[Brettanomyces secondary fermentation experiment]]

===External Resources===
* [http://www.homebrewtalk.com/funk-in-the-house-part-ii.html ''Funk In the House'', by Andrew J. Kazanovicz on Homebrewtalk].  Sensory analysis of 100% ''Brettanomyces'' fermentations of many of the commercially available strains.
* [http://www.bear-flavored.com/2015/06/what-is-brett-ipa-supposed-to-taste-like.html" What is Brett IPA Supposed to Taste Like."  Bear Flavored Blog.  Derek Dellinger.] - A great general description of what 100% ''Brettanomyces'' IPA tastes like.

==References==
<references/>

[[Category:Techniques]]