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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 because they lack lactic acid and 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]].

After primary fermentation, the mostly attenuated beer is sometimes moved to a secondary fermentation vessel (and sometimes not; read below). 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 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]).