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'''Mixed fermentation''' (also referred to as "mixed culture fermentation" or more specifically "multiple species mixed fermentation") is any fermentation that consists of a combination of ''[[Saccharomyces]]'' (brewer's yeast), ''[[Brettanomyces]]'' (wild yeast), ''[[Lactobacillus]]'' (lactic acid bacteria), and ''[[Pediococcus]]'' (lactic acid bacteria), or other [[Nonconventional_Yeasts_and_Bacteria|microbes that are unconventional in brewing]]. Broadly speaking, there are two styles of mixed fermentations: mixed fermentations with lactic acid bacteria (''Lactobacillus'' and/or ''Pediococcus'') and mixed fermentations without lactic acid bacteria. Mixed fermentation sour beers are characterized by their higher acidity and tart flavor caused by the production of [[Lactic Acid|lactic acid]], and require the use of a lactic acid bacteria (abbreviated as '''LAB'''; generally ''Lactobacillus'' and/or ''Pediococcus''). These beers generally fall within a pH range of 3.0-3.7 (although [[Titratable Acidity]] is more accurate for measuring perceived sourness). Mixed fermentation without lactic acid bacteria are usually fermented with a combination of ''Saccharomyces'' and ''Brettanomyces''. Mixed fermentation beers without lactic acid bacteria may be slightly tart from the [[Acetic Acid|acetic acid]] production of ''Brettanomyces'', but are generally not considered to be sour if well brewed becaues because they lack lactic acidand too much acetic acid is considered a flaw. For both categories, the primary fermentation will be completed by yeasts such as ''Saccharomyces'' and/or ''Brettanomyces''.

This page will focus on information for mixed fermentation sour beers using pure laboratory culturesand where the lactic acid bacteria is allowed to co-exist with yeast (e.g. not [[Wort_Souring#Souring_in_the_Boiler_.28Kettle_Sour.29|Kettle Sours]]). For mixed fermentation beers without lactic acid bacteria, see the [[Brettanomyces and Saccharomyces Co-fermentation]] page. For 100% ''Brettanomyces'' fermentations (technically not a "mixed" fermentation), see the [[100% Brettanomyces Fermentation]] page. 100% ''Lactobacillus'' or ''Pediococcus'' beers do not exist because they do not fully attenuate wort (see [[Lactobacillus#100.25_Lactobacillus_Fermentation|100% ''Lactobacillus'' fermentation]] for details). Other alternative yeast and bacteria can also be used, however this is currently not common even for brewers who make wild/sour beers. For example, [[Spontaneous Fermentation|spontaneous fermentation]] and [[Wild_Yeast_Isolation#Growing_and_Testing_Without_Plating|wild yeast captures]] usually contain a plethora of [[Nonconventional_Yeasts_and_Bacteria|yeast and bacteria that are not conventional to modern brewing]].

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 strain or a saison/farmhouse strain (see ''[[Saccharomyces]]'' page for a comprehensive list) to increase the ester and/or phenol characters of the beer which can then be acted on by ''[[Brettanomyces]]''. Primary fermentation with ''Saccharomyces'' also tends to lend to more glycerol production which increases the beer's mouthfeel (''Brettanomyces'' generally does not produce much glycerol <ref>[http://www.milkthefunk.com/wiki/Brettanomyces#Secondary_Metabolites Brettanomyces; Secondary Metabolites. MTF Wiki. Retrieved 06/23/2016]</ref>). 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>.This primary fermentation can take place in any vessel suitable for a normal ''Saccharomyces'' fermentation. As always In general, it is best practice to maintain fermentation temperature control is of critical importance and temperature profiles for this fermentation step should match those as suggested by the yeast lab for the strain of ''Saccharomyces'' selected for this step, although strict temperature control might not be completely necessary as long as the primary fermentation remains within the suggested temperature range of the selected ''Saccharomyces'' strain (the goal is to avoid off-flavor production from the ''Saccharomyces'' fermentation, although a higher amount of esters might be desirable and ''Brettanomyces'' can clean up some off-flavors like diacetyl in small amounts). 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 unsettled 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>. Concerns of yeast autolysis, however, have generally been minimized by most brewers (see [[Mixed_Fermentation#Secondary_Fermentation|Secondary Fermentation]]).

After primary fermentation, the mostly attenuated beer is sometimes moved to a secondary fermentation vessel(and sometimes not; read below). Often in traditional In commercial production secondary fermentation is often conducted in wine barrels(mostly because it is messy to conduct primary fermentation in 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 then these microbes 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 bottled 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. This also corresponds with a decrease in ''S. cerevisiae'' cell counts, and the release of amino acids and vitamins from yeast autolysis might help fuel helps feed lactic acid bacteria and ''Brettanomyces'' <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/1407620505932826/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. Other flavor-impacting secondary metabolites are also produced, depending on the strains used. For example, if the beer contains ''Brettanomyces'' this often results in the production of a high amount of fruity esters such as ethyl acetate and ethyl lactate, as well as "funky" phenols and other flavor compounds specific to ''Brettanomyces'' (see [[Brettanomyces#Secondary_Metabolites|''Brettanomyces'' secondary metabolites)]]. In the presence of oxygen, acetic acid is also produced by ''Brettanomyces'' (and acetic acid bacteria if they are present) which in low amounts can be complementary, adding to the complexity of the beer. In one study on mixed fermentation sour beer with one strain each of ''L. brevis'', ''B. bruxellensis'', and S-04, researchers found that diacetyl that was formed around month 2 had disappeared after another 4 months of aging, indicating that diacetyl, if present in earlier stages of fermentation, can age out of mixed fermentation beer. They also reported that the antioxidant phenol, guaiacol, was present above flavor threshold during all stages of aging from 2-12 months, and [[Isovaleric Acid]] was formed after 12 months of aging <ref>[https://www.mdpi.com/2076-2607/11/7/1681 Postigo, V.; García, M.; Arroyo, T. Study of a First Approach to the Controlled Fermentation for Lambic Beer Production. Microorganisms 2023, 11, 1681. https://doi.org/10.3390/microorganisms11071681.]</ref>. These flavor compounds are essential to the flavor profile of mixed fermentation sour beer. For example, traditional Berliner Weisse was fermented with a mixed culture containing ''Brettanomyces'', and this was considered the most important aspect of achieving the fruity ester character of that beer style historically (see [https://docs.google.com/spreadsheets/d/1CNrO46TPSFpjhO3HX1-CbKK5rhFd7uGWdpONf7AJAlU/edit#gid=0 Benedikt Koch's table comparing esters of traditional Berliner Weisse versus kettle soured Kindl Weisse and Belgian gueuze]).

Some brewers (including homebrewers and professional brewers) do not find it necessary to move the mostly attenuated beer into from the primary fermentation vessel to a secondary aging vessel. Instead, the mixed culture is pitched directly into the primary fermenter. While yeast autolysis is a concern in regular brewing, it is arguably 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 and then ethyl acetate production), and might be the best option if the brewer does not have a closed/CO2 system to prevent exposure to oxygen during transferring. Some [[Brettanomyces#Nitrogen_Metabolism|evidence suggests]] that the nutrients released by yeast autolysis are beneficial to ''Brettanomyces'', so leaving the beer on the yeast cake might even be more desirable than not. Some sour beer brewers strive to achieve autolysis in their beers with the belief that it could improve mouthfeel and react with other compounds to produce favorable flavors, similar to how autolysis is sometimes desired in winemaking in the form of [https://www.thekitchn.com/wine-words-lees-aging-179813 lees aging] or [https://www.thekitchn.com/wine-words-btonnage-191331 bâtonnage ]<ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3177292242298968/ Lars Meiner, Richard Preiss, and Alex Seitz. milk The Funk Facebook group thread on autolysis. 01/03/2019.]</ref>.

Sour beer should be aged in an environment that minimizes high temperatures and exposure to oxygen. Avoid temperatures over 85°F (29.5°C) and under 55°F (13°C). 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'', and these off-flavor metabolites are considered permanent. Filling the carboy to the neck or topping up carboys or barrels after primary fermentation will also help minimize the surface area of the beer that can be exposed to air. Topping up and flushing with CO₂ might also help reduce the risk of mold growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2640657275962470/?comment_id=2641402822554582&reply_comment_id=2642128842481980&comment_tracking=%7B%22tn%22%3A%22R%22%7D Lars Meiner. Milk The Funk Facebook group thread on preventing mold growth on krausen. 05/02/2019.]</ref> on any krausen material that has dried on the sides of the fermentation vessel after primary fermentation. Avoid oversampling the beer (once every 3 months at the very most). Using an "S airlock" has the benefit of showing if there is positive, negative, or equalized pressure in the fermenter, which could possibly assist in showing whether suck-back is a problem (see the Mark Trent YouTube video below). One way ventilated silicone bungs can be used for barrels or other waterless type airlocks (such as the [http://www.better-bottle.com/products_master.html BetterBottle "DryTrap"] or kegs with a [http://seanterrill.com/2015/06/25/build-a-better-spunding-valve/ spunding valve]; see also [[Sanke Fermenter]]) that allow gases to escape the fermenter but not enter from the environment, and [https://wwwbeer.redditthegremlyn.com/r2019/Homebrewing09/comments04/beolwvgrems-the-gas-reducing-multivessel-system/spunding_system_for_my_sour_program/ this Reddit post on Colin Burton's homebrew setup for connecting multiple cornelius kegs to a daisy-chained single spunding valve with corny kegs]. Topping up barrels with fresh beer every 3-6 months might help reduce acetic acid and ethyl acetate, and humidity and temperature control can help reduce evaporation (see [[Barrel#Using_Barrels_for_fermentation_and.2For_aging|Barrel]]). 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 overexposure to oxygen using water-based airlocks. For example, a small amount of oxygen helps [[Brettanomyces]] growth, and a small level of acetic acid is desirable for the complexity of long-aged sour beers <ref>[https://www.milkthefunk.live/podcast Richard Preiss. Interview on Milk The Funk "The Podcast" Episode #000. 12/13/2017.]</ref> (~30 minutes in). Higher levels of acetic acid are sometimes desirable for [[Flanders Red Ale]] style beers.

Headspace and fermenter size are also concerns when it comes to aging beer with living ''Brettanomyces''. This includes sour beers, [[Brettanomyces_and_Saccharomyces_Co-fermentation|non-sour beers with ''Saccharomyces'' and ''Brettanomyces'']], and [[100%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] that are aged. The larger the headspace, the more air will be sucked in when a vacuum occurs. The smaller the fermenter, the more headspace becomes a problem. Smaller vessels, in general, have a larger surface area to volume ratio. Therefore, they have more potential for exposure to oxygen. A large headspace in a smaller vessel exacerbates this problem, therefore it is advised to top up small fermenters and flush them with CO<sub>2</sub?> after primary fermentation or if significant evaporation occurs during aging. For example, a 1-gallon jug should be filled all the way to the neck if possible. A 5-gallon carboy could also be filled to the neck, but a little more headspace is permissible since it is a larger volume. Barrels are porous and the liquid inside them slowly evaporates. Some brewers combat this by topping up their barrels on a regular basis; this also helps keep the top staves from drying out (higher humidity can help limit evaporation; see the [[Barrel#Using_Barrels_for_fermentation_and.2For_aging|Barrel]] page).

One misconception about aging beers is the claim that CO₂ is heavier than air and forms a blanket that protects the beer from oxygen. This is not true unless CO₂ is constantly being produced from the beer. The [https://en.wikipedia.org/wiki/Ideal_gas_law Ideal Gas Law] states that unlike solids or liquids of different densities, the gasses will of different densities eventually mix. See [http://beerandwinejournal.com/can-co2-form-a-blanket/ Dr. Chris Colby's explanation of this on Beer and Wine Journal.], and this [https://www.youtube.com/watch?v=_oLPBnhOCjM science video documentary demonstration of how gasses eventually mix] (note that the molecular weight of bromine used in the video is 160 g/mol and the weight of CO₂ is 44.01 g/mol, so CO₂ would diffuse into air faster than bromine <ref>[https://pubchem.ncbi.nlm.nih.gov/compound/Dibromine Bromine. PubChem. Retrieved 1/1/2016.]</ref><ref>[https://pubchem.ncbi.nlm.nih.gov/compound/280 Carbon Dioxide. PubChem. Retrieved 1/1/2017.]</ref>).

Methods of creating sour beer without using ''Brettanomyces'' are also considered a form of mixed fermentation. In general, these methods include pitching a pure culture of ''Lactobacillus'' along with brewers yeast at the same time or staggered with pitching ''Lactobacillus'' first for a day or two and then brewers yeast (Cascade Brewing is known for the latter process <ref>Tonsmeire, Michael. "American Sour Beers: Innovative Techniques for Mixed Fermentations". Brewers Publications, Jun 15, 2014. Pg 125.</ref>). In some cases, the brewer's yeast (''Saccharomyces cerevisiae'' or ''Saccharomyces pastorianus'') can be pitched first, and then the ''Lactobacillus'' is pitched (see "Reverse MTF Method" below). Since ''Brettanomyces'' is removed from the process, these methods tend to create a sour beer in a shorter amount of time, but without the complex ester and phenol profile of ''Brettanomyces''. While these types of beers may be less complex than beers with ''Brettanomyces'', they produce a different beer than [[Wort Souring|Kettle soured]] beers. Several studies have shown that co-fermentation of brewers yeast and lactic acid bacteria produces an objectively different beer than kettle souring or adding pure lactic acid. Pre-acidifying with lactic acid bacteria fermentation can also negatively affect the primary yeast fermentation, but other studies have shown that it can also result in a faster fermentation time but with less attenuation and other methods less yeast growth. See the [[Lactobacillus#Effects_on_Mixed_Fermentation|''Lactobacillus'' Effects on mixed Fermentation]] wikipage for more information on these studies.  ===="Reverse MTF Method"====Devin Bell reported getting a good level of sourness by co-pitching probiotics with ''L. plantarum'' or Omega Labs OYL-605 with yeast, or even after primary fermentation (also known colloquially as the "Reverse MTF Method"). By allowing the yeast to ferment for two or three days before adding ''Lactobacillus'' for souring wort before , it is claimed that this method allows the yeast character to be expressed more so than with kettle sours. In the case of pitching brewer's 'L. plantarum'' after fermentation with saison yeast, Bell reported that the beer turned out like a sour saison, where as well as pitching lactic co-pitched makes for a better Berliner Weisse or Gose style beer without the "saison" yeast character. This has also improved head retention in his beers. Using no hops seems to be required in order to get acid bacteria production from the ''L. plantarum'' after primary fermentation . Devin clarified that his "best success" is pitching ''S. cerevisiae'' saison strain with brewera selection of ''Brettanomyces''s yeastfor primary fermentation. After 5-7 days of fermentation, are examples he pitches ''L. plantarum'' (2 shots of GoodBelly or 1 package of Omega Labs OYL-605 for 5-6 gallons of this processbeer) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1739156616112545/?comment_id=1739183316109875&reply_comment_id=1739749539386586&comment_tracking=%7B%22tn%22%3A%22R%22%7D Devin Bell. Milk The FunkFacebook group. 06/30/2017.]</ref>. Once terminal gravity is reached (1.002-1.004), he bottles right away. The bottles can be served at 8 weeks in the bottle, but start to peak at 24 weeks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1562696320425243/?comment_id=1562702310424644&comment_tracking=%7B%22tn%22%3A%22R0%22%7D MTF discussion with Devin Bell on "reverse MTF kettle sour. 01/26/2017.]</ref>. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1217518078276404/ this thread by Devin Bell] and [Wort Souringhttps://www.facebook.com/groups/MilkTheFunk/permalink/1728489143845959/ this thread by (Zach) Caroline Whalen Taggart]. See also:* [https://www.facebook.com/SouthernBrewersConference/videos/881235365357984/ Devin Bell and Dan Pixley presentation at SBC 2017 on quick souring, including more details on this method.] and * [[Alternative_Bacteria_Sources#MTF_Culturing_from_Probiotics|General tips on using probiotics to make sour beer]].22Reverse_Kettle_Sour ==Finishing Mixed Fermentation Sour Beer=====Determining When It Is Done===Unless the brewer has worked with the same blend of microbes and wort recipe, it is difficult to give an exact time frame on when a mixed fermentation beer might be ready. Anywhere from three to twelve months (and sometimes longer) is a reasonable amount of time, but when any given beer will be ready for packaging within this time depends on many factors including the microbes pitched, their health over time, wort composition, temperatures during the aging time, etc.22|MTF "Reverse Kettle Sour" The best guide is a long-term stable gravity: if the beer's gravity has remained stable between several readings over a month or two, then the beer may be ready for packaging. The second factor is how does the beer taste? If it tastes good, and the gravity is stable, then it can be packaged. If the beer does not seem to have a mature flavor or has off-flavors that need to age out, then feel free to age it longer. Some off-flavors will change even when bottled or kegged, but others (such as sulfur-based compounds) will need to dissipate out of the fermenter slowly over time. ===Bottling and Kegging===See the [[Packaging]]page.