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For the purposes of this article, we are defining a '''mixed fermentation''' as any fermentation that consists of a combination of ''[[Saccharomyces]]'', ''[[Brettanomyces]]'', ''[[Lactobacillus]]'', and ''[[Pediococcus]] '' cultures. Broadly speaking, there are two styles of mixed fermentations: "sour" fermentations and "funky" fermentations. "Sour" fermentations are characterized by their higher acidity and tart flavor, and require the use of a Lactic Acid Bacteria (LAB; generally ''Lactobacillus '' and/or ''Pediococcus''). "Funky" fermentations generally do not contain LAB, but instead use a combination of ''Saccharomyces '' and ''Brettanomyces''. Funky beers may be slightly tart, but are generally not considered sour. For both categories, the primary fermentation will be completed by yeasts such as ''Saccharomyces '' and/or ''Brettanomyces''. For ''Funky Mixed Fermentations'', see the [[Funky Mixed Fermentations]] page. For 100% ''Brettanomyces '' fermentations, see the [[100% Brettanomyces Fermentation]] page.

==''"Sour'' " Mixed Fermentation - The Basics==Sour fermentations require at least one Lactic Acid Bacteria (LAB), such as ''[[Lactobacillus]] '' or ''[[Pediococcus]]'', and at least one yeast such as ''[[Saccharomyces]] '' or [[Brettanomyces]]. Many yeast companies offer [[Mixed Cultures]] that provide all of the microorganisms necessary to make a sour beer. The results of these commercial mixed cultures can be as varied as the cultures themselves. For example, some of these commercial mixed cultures produce lightly tart beer that may exhibit minimal funky flavors; others may produce intense sourness and assertive funk. This is dependent on the types of microbes in the mixed culture, their ratios, how old the cultures are, and what methods the brewer uses to encourage or discourage certain flavors. The brewer must understand that all of these microbes are complex organisms (some more complex than others). Not only do different species behave differently and produce different results under different conditions, but different strains of the same species also can also behave differently and produce different results under different conditions. Just as strains of ''Saccharomyces cerevisiae '' produce different results in clean beers (e.g., California Ale yeast versus Belgian Ale yeast), strains of ''Lactobacillus '' spp. and especially ''Brettanomyces '' spp. can also vary widely.

The most basic method for making a mixed fermentation sour beer is to brew some simple wort (fresh extract or all grain) that is low in IBU's. Iso-alpha acids can inhibit many species and strains of LAB. Keeping the wort less than 6 IBU's is recommended in general, unless the brewer has information about their LAB culture that indicates that they can tolerate more. Mash hopping is one technique that can be used to limit the IBU's by about 70% <ref>[http://www.homebrewersassociation.org/attachments/presentations/pdf/2014/Putting%20Some%20Numbers%20on%20First%20Wort%20and%20Mash%20Hop%20additions.pdf Putting Some Numbers on First Wort and Mash Hop additions. David Curtis. NHC 2014.]</ref>. If hops are not required (commercial brewers may be required to use hops, while homebrewers aren't), they can be completely excluded from the recipe. The wort is often mashed at a high temperature to encourage the inclusion of complex carbohydrates in the final wort. The wort is then Primary fermented with a ''Saccharomyces '' strain to achieve the majority of attenuation, leaving behind the complex carbohydrates. The primary fermentation is then inoculated with a mixed culture of ''Brettanomyces'', ''Lactobacillus '' and ''Pediococcus'', either by moving the wort into barrels with active cultures or by inoculating the primary fermentation vessel (i.e. glass carboy when the method is used by home brewers). This inoculation then starts a secondary fermentation of the remaining complex carbohydrates which follows a slow progression between the microbes that are primarily active. This secondary fermentation may not show readily apparent sighs of active fermentation as in the primary fermentation but is often accompanied by the slow evolution of CO2 in the first 8 weeks and the eventual formation of a pellicle which may form quickly or very slowly.

The grain bill and production for the wort doesn't have to be complex, in fact many sour breweries produce their full line of sours from 2-3 base sour recipes which are then modified after aging by blending, the addition of fruit, dry hops or simply packaging them without alteration. For sour blonde ales, a simple grain bill of about 70% Pilsner malt and 30% malted wheat can be used (these can be replaced by Pilsner and wheat unhopped extracts for the extract brewer. See [http://byo.com/videos/item/975-lambic-brewing Lambic Brewing by Steve Piatz] or [http://www.homebrewtalk.com/showthread.php?t=322168 AmandaK's lambic-style extract recipe] for a good extract recipe). Some crystal and a small amount of roasted malts an be used for sour brown ales. Some higher chain sugars or even starches can be included for beers that will be aged for a long time and include ''Brettanomyces'', or ''Brettanomyces '' and ''Pediococcus '' ''(Pediococcus Pediococcu''s generally should not be used without ''Brettanomyces''. See the ''[[Pediococcus]] '' page for more details). Performing a [[Turbid Mash]] is the traditional way to include starches in the wort. However other methods such as steeping some oats or flaked wheat during the boil <ref>[http://www.homebrewtalk.com/f127/adjuncts-starches-sour-beer-448529/ Homebrewtalk Discussion started by Amos Brown aka 'Metic']</ref>, or running off over a bag of flaked oats or wheat on the way to the kettle can also impart starches that won't be converted to sugars by the mash (see [[Turbid_Mash#Alternative_methods_to_yield_starchy_wort|alternatives to turbid mashing]]). This step is completely optional, however it may be very beneficial to make sure some higher chain sugars or starches are available in the wort if the brewer wants to rely on ''Pediococcus '' for producing most of the acidity. Extract brewers can use 0.25 lbs. (0.11 kg) of Maltodextrin <ref>[http://byo.com/videos/item/975-lambic-brewing Lambic Brewing. Piatz, Steve. Brew Your Own Magazine. October, 2004.]</ref>, or hot steep a pound of flaked wheat, flaked oats, or carapils malt.

Questions often arise regarding Brewers have historically had concerns about aerating wort that has either been pre-soured with lactic acid bacteria (if the lactic acid bacteria is still alive) or if it will receive a co-pitch of lactic acid bacteria, ''[[Brettanomyces]]'', and when wort aeration should be done''[[Saccharomyces]]'' (see [[Mixed_Fermentation#Reusing_a_Sour_Yeast_Cake|Reusing a Sour Yeast Cake]], [[Mixed_Fermentation#Multi-Stage_Fermentation|Multi-Stage Fermentation]] and [[Sour Worting]]). It is well documented that These concerns, however, are largely unfounded. Most species of ''[[SaccharomycesLactobacillus]] uses '' are either not effected by oxygen to biosynthesize lipids, which include fatty acids and sterolsor benefit slightly. [[Butyric Acid]] production by ''Lactobacillus'' is not a concern (see [[Lactobacillus#Effects_of_Oxygen|''Lactobacillus'', effects of oxygen]] for their cell membranesdetails). The cell membrane regulates the flow Some species/strains of nutrients into ''[[Pediococcus]]'' might be inhibited by oxygen, but not all (see ''[[Pediococcus#Growth_and_Environment|Pediococcus]]'' for details). ''[[Brettanomyces]]'' creates acetic acid in the cell and waste outside presence of the celloxygen, and allows however in the yeast to reproduce. Each time presence of a yeast cell doubles during growthhealthy pitch of ''[[Saccharomyces]]'', which rapidly consumes the parent cell gives approximately half of its lipids to the daughter celloxygen, this is probably also not a concern. The more sugar available to the yeastAdditionally, oxygen greatly improves the more they will reproduce, vitality and thus the more lipids they requirecell count of ''Brettanomyces'' (see [[Brettanomyces Propagation Experiment]]). Thus, without as long as a healthy cell membrane and a build up pitch of lipids, the cell can die or produce weak daughter cells, potentially resulting in a range of off-flavors, especially in higher gravity beers <ref name="Aquilla">''[[https://www.morebeer.com/articles/how_yeast_use_oxygen "The Biochemistry of Yeast," by Tracy Aquilla. Morebeer Website. 07/25/2013. Retrieved 04/13/2016.Saccharomyces]</ref><ref name="danstar">[http://www.danstaryeast.com/articles/aeration-and-starter-versus-wort Aeration And Starter Versus Wort. Danstar Website. Retrieved 04/13/2016.]</ref>. Therefore in the brewing of non-mixed fermentation beers'' is present, aerating both the yeast starter and the wort before pitching the yeast is generally considered mandatory for mixed fermentation should lead to the brewing processa healthy fermentation and good results.

Brewers have historically had concerns about aerating Many brewers, however, do not aerate their wort that has when either been pre-soured souring the wort with lactic acid bacteria (and is still alive) or will receive a co-pitch pure culture of lactic acid bacteria, ''[[Brettanomyces]]'', and ''[[Saccharomyces]]'' (see [[Mixed_Fermentation#Reusing_a_Sour_Yeast_Cake|Reusing pitching fresh wort on top of a Sour Yeast Cake]]mixed culture yeast cake, [[Mixed_Fermentation#Multico-Stage_Fermentation|Multi-Stage Fermentation]] and [[Sour Worting]]), however these concerns are largely unfounded. Most species of ''[[Lactobacillus]]'' are either not effected by oxygenpitching a mixed culture such as Wyeast Roeselare, or benefit slightly ([[Butyric Acid]] production by ''Lactobacillus'' is not pitching a concern; see custom mix of microbes from multiple sources <ref>[[Lactobacillus#Effects_of_Oxygen|''Lactobacillus'', effects of oxygen]] https://www.facebook.com/groups/MilkTheFunk/permalink/1182597671768445/?qa_ref=qd Conversation on MTF about oxygenating wort for details)mixed culture fermentation. Some species11/strains of ''[[Pediococcus22/2015.]]'' might be inhibited by oxygen, but not all (see ''[[Pediococcus#Growth_and_Environment|Pediococcus]]'' for details)</ref>. Many such brewers have reported success without aerating. ''[[Brettanomyces]]'' creates acetic acid from oxygen, however We therefore recommend that the brewer investigate and experiment with their process in the presence of a healthy pitch of ''[[Saccharomyces]]'', which rapidly consumes the oxygen, this is probably also order to decide whether or not a concern. Thus, as long as a healthy pitch of ''[[Saccharomyces]]'' aeration is present, aerating wort for mixed fermentation should lead to a healthy fermentation and good resultsdesired.

Many brewers, however, do not aerate their wort when either pre-souring the wort with a pure culture of lactic acid bacteria, pitching fresh wort on top of a mixed culture yeast cake, co-pitching a mixed culture such as Wyeast Roeselare, East Coast Yeast Bug County, or pitching a custom mix of microbes from multiple sources <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1182597671768445/?qa_ref=qd Conversation on MTF about oxygenating wort for mixed culture fermentation. 11/22/2015.]</ref>. Many such brewers have reported success without aerating. We therefore recommend that the brewer investigate and experiment with their process in order to decide whether or not aeration is desired.  If the brewer is pitching a separate liquid culture of ''[[Saccharomyces]]'', it is recommended to create a starter on a stir plate and alternatively doze dose it with oxygen. This will allow the cell membranes to build enough lipids for their cells walls and give them the greatest chance of fermenting the wort without off-flavors <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1284106178284260/?comment_id=1284141108280767&comment_tracking=%7B%22tn%22%3A%22R3%22%7DConversation with Mark Trent on MTF regarding aerating starters/wort for mixed culture fermentations. 04/13/2016.]</ref>. If the wort has been pre-soured, it might be beneficial to propogate the yeast starter with a portion of the soured wort equal to the portion of starter wort in order to acclimate the yeast to the has conditions (see [[Saccharomyces#Fermentation_Under_Low_pH_Conditions|''Saccharomyces'' fermentation under low pH conditions]]). Dried yeast is grown and processed in such a way that they contain enough lipids to support a healthy fermentation of 5% ABV or less without the need for aeration (this may be dependent on manufacturer; see the yeast manufacturer's website for their individual recommendations). Above 5% ABV however, and aeration Aeration should be considered for beers above 5% <ref name="danstar"></ref>. (To do - starters for mixed cultures)

Primary Fermentation by ''Saccharomyces '' is generally conducted in the same way for a sour beer as for a non-sour beer. Depending on the intended final result the brewer might select a neutral ale strain (WLP 001-Wyeast 1056, WLP036-Wyeast 1007) to provide a neutral background for the souring microbes to act on. Alternatively, the brewer may use a belgian-farmhouse strain (see ''[[Saccharomyces]] '' page for a comprehensive list) to increase the phenol characters of the beer which can then be acted on by ''[[Brettanomyces]]''. This primary fermentation can take place in any vessel suitable for a normal ''Saccharomyces '' fermentation, as always fermentation temperature control is of critical importance and temperature profiles for this fermentation step should match those suggested for the strain of ''Saccharomyces '' selected for this step. Once active fermentation has subsided the mostly attenuated wort can then be moved on to the secondary fermenting vessel. There is some variation in common practice as to whether or not the primary fermentation yeast should be carefully settled out, moving over bright clear beer only, or if un-settled cloudy high yeast population wort is moved to the secondary vessel. New Belgium moves their lager primary fermented beer after centrifuging, indicating that this centrifuged beer exhibits cleaner characters from secondary fermentation faster than un-centrifuged beer, allowing the resulting sour beer to be ready for packaging more quickly <ref> The Sour Hour Episode 2 with Lauren Salazar from New Belgium Brewing Company ]</ref>.

After primary fermentation the mostly attenuated beer is moved to a secondary fermentation vessel. Often in traditional commercial production secondary fermentation is conducted in wine barrels, however home brewers can accomplish this phase in glass or plastic carboys with low oxygen permeability. A mixed culture of ''Brettanomyces'', ''Lactobacillus '' and ''Pediococcus '' is then introduced to the beer. If barrels are being used these "bugs" may simply come from the walls of the barrel, originating from a previous batch. Alternatively, the brewer might inoculate the wort with a mixed culture directly, either with a house culture or by introducing the dregs of sour beer. Upon their introduction these new microorganisms begin converting the longer chain sugars left over from the primary fermentation. These sugars are primarily converted into alcohol and lactic acid, increasing the degree of attenuation and lowering the pH of the beer. In the presence of oxygen acetic acid is also produced which in low amounts can be complementary, adding to the complexity of the beer.

Some brewers (mostly homebrewers) do not find it necessary to move the mostly attenuated beer into a secondary vessel. Instead, the mixed culture is pitched directly into the primary fermenter. While yeast autolysis is a concern in regular brewing, it is less so not a cause for concern in mixed fermentations that contain ''Brettanomyces''. Lambic brewers, for example, perform a primary fermentation in barrels, and leave the beer in the barrels during the beer's entire aging process, which is usually 1-3 years <ref>[http://www.lambic.info/Brewing_Lambic#Barrels Lambic.info Wiki. Brewing Lambic. Retrieved 6/8/2015.]</ref>. Yeast autolysis releases trehelose, acids, and other compounds, which are metabolized by ''Brettanomyces '' <ref>[http://www.mbaa.com/districts/michigan/events/Documents/2011_01_14BrettanomycesBrewing.pdf Brettanomyces in Brewing the horse the goat and the barnyard. Chad Yakobson. 1/14/2011.]</ref>. Maintaining a [[Solera]] may be an exception to this (see the [[Solera]] page for details). The advantage of not moving the beer into a secondary vessel is that less overall oxygen is introduced into the beer (oxygen exposure will contribute to more acetic acid production), and may be the best option if the brewer does not have a closed/CO2 system to prevent exposure to oxygen during transferring.

Aging is generally required for mixed fermentations that include ''Brettanomyces''. The necessary/ideal amount of aging time will depend on the microbes pitched, the pitching rate, and the desired final beer. Keep in mind that the beer will also continue to develop once packaged. For more straightforward beers with highly attenuative primary strains (like tart saisons), a reasonable final product with tartness and brett ''Brettanomyces'' character can be reached in a few months. For more complex and/or acidic beers (such as Flemish reds or beers inspired by lambics) you may expect an aging time of at least 9 months, but quite possibly as long as 12-18 months or longer. In general longer aging will allow more complex expression of the spectrum microbes present.

Sour beer should be aged in an environment that minimizes high temperatures and exposure to oxygen. Drastic temperature fluctuations and changes in atmospheric pressure will cause a vacuum inside of the fermentation vessel causing water airlocks to "suck back" air into the fermenter. This could potentially contribute to [[Acetic Acid]] and [[Ethyl acetate]] (nail polish aroma in high concentrations) production by ''Brettanomyces'', however the development of a pellicle can help protect against this. Filling the carboy to the neck will also help minimize the surface area of the beer that can be exposed to air. Avoid over sampling the beer (once every 3 months at the very most). It should also be noted that micro-oxygenation is helpful for creating certain flavors in sour beer, and many homebrewers have reported not having any issues with over exposure to oxygen using water-based airlocks.

# Pitch a pure culture of ''[[Lactobacillus]]'', and if possible hold the temperature between 110-120°F (43.3-48.9°C) for 2-4 days (see the [[Sour_Worting#Souring_in_the_Primary_Fermenter|Souring in the Primary Fermenter]] page for more details).# After 2-4 days, cool the wort to 65-70°F (18.3-21.1°C), oxygenate the wort, and pitch a starter of ''[[Saccharomyces]]''.

# Add one or more cultures of ''[[Brettanomyces]]''. Optionally, also pitch a culture of ''[[Pediococcus]] '' and/or bottle dregs from commercial sours (see [[Commercial_Sour_Beer_Inoculation|Commercial Sour Beer Inoculation]] for more details on using commercial bottle dregs). For more ''funky'' Brett flavors, do not make a starter for the Brett. '''(Editor's note: new information suggests that pitching rate for ''Brettanomyces '' in a mixed fermentation probably does not impact flavor. See [[Brettanomyces secondary fermentation experiment]] for more details).'''

Reusing a sour yeast cake can often provide great results. Brewers have reported success repitching re-pitching on very old yeast cakes (2 years) without getting off flavors from yeast autolysis. After several months, ''[[Saccharomyces]] '' tends to die off due to the low pH in a sour beer. The bacteria and [[Brettanomyces]] tend to survive the lower pH, and their cell counts can be high in even an old yeast cake. By pitching new wort on an old sour yeast cake, these microbes (particularly the [[Lactobacillus]]) have access to the simple sugars in the wort <ref>[http://www.themadfermentationist.com/2009/11/brewing-sour-beer-at-home.html Tonsmeire, Michael. The Mad Fermentationist. Brewing Sour Beer at Home. Last paragraph in the "Inoculation" section. Retrieved 2/19/2015.]</ref>. In general, washing the yeast cake is not necessary.

Some brewers have good success reusing a yeast cake or a portion of a yeast cake by leaving the wort in contact with the old yeast cake for 1-4 days. After 1-4 days, a fresh culture of ''Saccharomyces '' is pitched to finish the fermentation. The 1-4 day head start gives the souring bacteria a head start and results in a low pH beer. The harvested yeast cake can also be pitched at the same time as a fresh culture of Saccharomyces, or afterwards. The decided timing on when to pitch the harvest sour yeast cake will effect the outcome of the sour beer. Early pitching of the sour yeast cake generally produces a more acidic beer, and later pitching generally produces a less acidic beer.

As with all methods, the species and strains of the microbes being used should always be taken into consideration. Experimentation and repeated processes should be carefully employed by the brewer in order to find the best results for their cultures. For example, using different strains of ''Saccharomyces cerevisiae '' as the primary fermenter can produce widely different results.