Mixed Fermentation

This article is about sour brewing methods using commercial cultures. For other brewing methods, see Brewing Methods.

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 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, 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 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 cultures and where the lactic acid bacteria is allowed to co-exist with yeast (e.g. not 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 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 and wild yeast captures usually contain a plethora of yeast and bacteria that are not conventional to modern brewing.

It is important to mention that mixed fermentation brewing in general has very few well-established rules and definitions. While we may categorize techniques for the sake of keeping some sort of manageable structure to this wiki, many methods can be used in conjunction with other Brewing Methods, and brewers sometimes use same/different terminology for the same/different things (for example, the use of the term "wild beer" by professional brewers can mean "any mixed fermentation beer", or can also mean "mixed fermentation beer brewed with wild caught microbes"). New methodologies are constantly being developed that combine elements of more established techniques, as well as slight changes to established techniques. Definitions equally evolve over time. Many of the methods used are determined by the types of microbes the brewer is working with. An article of this length cannot encompass all mixed fermentation methods. Instead it will provide a "big picture" view of the general methodologies. Towards this end, we divide mixed fermentation methods into two approaches: the traditional long fermentation method and an increasingly popular, short fermentation method. They are divided here as a device to illustrate the philosophy of each and facilitate the discussion of the techniques used for each methodology. The distinction of these two methods is however somewhat artificial, indeed many brewers use elements of both approaches to achieve their desired results. Examples of how techniques can overlap to create new techniques can also be found in Michael Tonsmeire's pivotal book on mixed fermentation brewing, "American Sour Beers". An archive of various sour beer terminology discussions and debates can be found here.

Mixed Fermentation Sour Beer - 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.

BJCP styles that can be brewed using this method include Berliner Weissbier and the subcategories of American Wild Ale, which include Mixed-Fermentation Sour Beer and Soured Fruit Beer . Belgian sour styles such as Lambic, Gueuze, and Fruit Lambic, technically can only be produced by Spontaneous Fermentation, however for beer competitions process is less important than what the resulting beer tastes like. Flanders Red Ale and Oud Bruin styles can be brewed using pure cultures, but can also be brewed using spontaneous fermentation or a mix of using pure cultures and spontaneous fermentation.

Traditional Method - Long Fermentation

Introduction

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% . 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 readily show apparent signs 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 depending on the presence of oxygen. This method is still the most commonly used by commercial producers of modern and traditional Belgian sour beer, with variations on the process occurring widely. While still widely used by homes brewers, fast fermentation methods such as Wort Souring and other methods mentioned in this article are ever increasing in their use.

Wort Production

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 Lambic Brewing by Steve Piatz or 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 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 , 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 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 , or hot steep a pound of flaked wheat, flaked oats, or carapils malt. See MTF Member Recipes for ideas on recipes, or the recipe sections of the books "American Sour Beers" by Michael Tonsmeire and "Wild Brews" by Jeff Sparrows.

Aeration

Questions often arise regarding if and when wort aeration should be done. It is well documented that Saccharomyces uses oxygen to biosynthesize lipids, which include fatty acids and sterols, for their cell membranes. The cell membrane regulates the flow of nutrients into the cell and waste out of the cell, and allows the yeast to reproduce. Each time a yeast cell doubles during growth, the parent cell gives approximately half of its lipids to the daughter cell. The more sugar available to the yeast, the more they will reproduce, and thus the more lipids they require. Therefore, without a healthy cell membrane and a build up of lipids, the cell can die or produce weak daughter cells, potentially resulting in a range of off-flavors, especially in higher gravity beers . In the brewing of non-mixed fermentation beers, aerating both the yeast starter and the wort before pitching the yeast is generally considered mandatory to the brewing process.

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 Saccharomyces (see Reusing a Sour Yeast Cake, Multi-Stage Fermentation and Wort Souring). These concerns, however, are largely unfounded. Most species of Lactobacillus are either not affected by oxygen, or benefit slightly. Butyric Acid production by Lactobacillus is not a concern (see Lactobacillus, effects of oxygen for details). Some species/strains of Pediococcus might be inhibited by oxygen, but not all (see Pediococcus for details). Brettanomyces creates acetic acid in the presence of oxygen, however, in the presence of a healthy pitch of Saccharomyces, which rapidly consumes the oxygen, this is probably also not a concern. Additionally, oxygen greatly improves the vitality and cell count of Brettanomyces and a small amount is required for effective Brettanomyces growth and fermentation (see Brettanomyces Propagation Experiment). Thus, as long as a healthy pitch of Saccharomyces is present, aerating wort for mixed fermentation should lead to a healthy fermentation and good results.

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, or pitching a custom mix of microbes from multiple sources . 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 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 . If the wort has been pre-soured, it might be beneficial to propagate 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). 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). Aeration should be considered for beers above 5% . Mixed cultures can also benefit from a starter if they are expired or haven't been stored correctly (see mixed culture starters).

Microbe Inoculation

Once the wort is produced and chilled, the mixed culture can be pitched as normal. If using a mixed culture from a commercial yeast lab, a starter is generally not needed. If the culture is old or a larger volume is needed, generally a normal starter can be made for mixed cultures without fear of "throwing off the balance of microbes" (see The Yeast Bay starter tips as an example; Bootleg Biology and Omega Yeast Labs also recommend starters for mixed cultures for larger batches ). Instead of buying a single mixed culture, a brewer can create their own mixed culture by combining their own ratios from single cultures of yeast and bacteria. A single mixed culture can be supplemented by adding pure cultures from different yeast labs as well. Another suggestion that often helps produce a higher quality sour beer is to supplement the mixed culture with a Commercial Sour Beer Inoculation. In general, the more diversity of microbes, the more complex a sour beer can potentially be. Using a Wort Souring method in conjunction with this method can help increase acidity that may not otherwise be produced by some commercial blends (Wyeast Roeselare is known for this characteristic, for example).

Staggered Versus Co-Pitching

Staggered pitching versus co-pitching can have a significant impact on the final flavor profile of the beer. While there is a lot of information regarding the fermentation profile of various microbes used in sour brewing, the impact of co-fermentation is less understood. Butler et al., partnered with Gilded Goat Brewing Company, analyzed the differences between co-pitching S. cerevisiae, a strain of B. bruxellensis, and a strain of L. plantarum (Sample A), versus pitching the S. cerevisiae and B. bruxellensis first and then the L. plantarum three days later (sample B), versus pitching the L. plantarum first and then the S. cerevisiae and B. bruxellensis three days later (Sample C). The three different beers were aged for a month and a half before packaged. Sample A was characterized as tasting the most balanced and consumers preferred it. Sample B was preferred the least and was characterized as having more "funk" flavor. Sample C had a distinctly sharp lactic sourness that overwhelmed the flavor from the Brettanomyces, despite having slightly less lactic acid and a slightly lower titratable acidity than Sample A. Each of the three different fermentation profiles had a different sensory fingerprint with different measurements for proteins, titratable acidity (slight differences), lactic acid (slight differences), polyphenols, turbidity, color, and residual sugar, indicating that when individual species are introduced to ferment the wort, that it potentially has a wide impact on many different aspects of the beer. See the full poster by Butler et al. here, as well as clarifications and corrections to the "Conclusion" statements in the poster by Charlie Hoxmeier of Gilded Goat Brewing Company . These results may or may not be repeatable with different strains or other variables, but it does demonstrate that co-pitching and staggered pitching produce measurably different results.

Depending on the ethanol tolerance of the lactic acid bacteria strains present in the culture, the presence of ethanol can have a negative impact on lactic acid bacteria. For example, in one published study, at 11% ABV, the strain of L. brevis used in the study didn't grow as well than as in lower ABV samples, and the resulting lactic acid content was lower as well ion the 11% ABV beer . Therefore, adding the lactic acid bacteria earlier in the fermentation process versus later in higher ABV beers will most likely impact the final beer's lactic acid content.

See also:

• Brülosophy blind triangle test between co-pitched vs staggered pitched mixed fermentation sour beer. See also the associated Brülosophy podcast episode.
• Multi-Stage Fermentation below.
• Cofermentation of Brettanonyces and Saccharomyces.
• Mixed Fermentation Without Brettanomyces.
• Effects of Lactobacillus on cofermentation.

Stages of Fermentation

Conceptual graph of traditional souring microbe and wort dynamics. Y-axis for each microbe group depicts relative activity which combines in a conceptual sense: growth, acidification of wort, attenuation and production of flavor compounds. Plot drawn by Drew Wham based on concepts discussed in American Sour Beer and Wild Brews .

Primary Fermentation

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 ). However, the role of glycerol in creating mouthfeel is debatable in the wine world . This primary fermentation can take place in any vessel suitable for a normal Saccharomyces fermentation. In general, it is best practice to maintain fermentation temperature control 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 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 . Concerns of yeast autolysis, however, have generally been minimized by most brewers (see Secondary Fermentation).

During both primary and secondary fermentation, a complex set of interactions occurs between the various yeast and bacteria species. Much of this is as yet unknown scientifically. For example, the production of lactic acid by lactic acid bacteria not only stresses and limits growth of S. cerevisiae, but it can also turn off "glucose repression", meaning that instead of consuming simple sugars first, S. cerevisiae stops choosing which sugar types to consume first and consumes all sugar types indiscriminately. This can result in under-attenuation problems in the short run, but also more residual sugars for Brettanomyces (see lactic acid for details). Another example is that co-fermenting with brewer's yeast and Lactobacillus can create a different flavor profile than if they are staggered with a kettle souring method (see effects of Lactobacillus on mixed fermentation). Another example is that some studies support that in nitrogen rich substrates, S. cerevisiae will synthesize simpler amino acids from the more complex nitrogen sources, and those amino acids contribute to the sustained survival of both Lactobacillus and Brettanomyces in the more stressful, post-fermentation environment (see also this MTF thread).

Secondary Fermentation

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 . 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). 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 . 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 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 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 . Yeast autolysis releases trehelose, acids, and other compounds, which are metabolized by Brettanomyces . 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 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 lees aging or bâtonnage .

Co-pitching all of the microbes to begin with, including the primary Saccharomyces culture, can produce different results than staggering the pitches of individual species over time. For example, many brewers pitch a single mixed culture that contains ale yeast, Brettanomyces, and lactic acid bacteria. Other brewers, such as Vinnie Cilurzo at Russian River, prefer to pitch their ale yeast first, and then pitch Brettanomyces and/or lactic acid bacteria after the primary fermentation . See this Brulosophy experiment comparing co-pitching versus staggered pitching (note that oxygen exposure during the staggered pitch and other variables in this experiment could account for some of the differences between the two beers) .

It is not unusual to see a slight rise in pH during the secondary or aging phases. For example, Santeri Tenhovirta's in his masters thesis he measured the pH of several species of Lactobacillus that were pitched into wort for 2 days, followed by US-05. Tenhovirta reported a slight pH rise of about 0.3 from day 150 until day 300-330. According to Kunze and Bamforth, an increase in pH towards the end of fermentation or aging could be caused by yeast autolysis .

Aging

Aging is generally required for mixed fermentations that include Brettanomyces. The necessary/ideal amount of aging time will depend on many factors including the microbes pitched, the pitching rate, wort composition, storage temperature, 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 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. Some brewers will package a beer after the finishing gravity has stabilized (see Packaging), and allow the beer to fully develop in the bottle. Keep in mind that some volatile flavor compounds, such as sulfur-based compounds, may volatilize off at a faster rate in a fermenter (especially a shallow fermenter such as a barrel) than they would in a sealed bottle, and bottling too early can result in over-carbonation.

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 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 BetterBottle "DryTrap" or kegs with a spunding valve; see also Sanke Fermenter) that allow gases to escape the fermenter but not enter from the environment, and Colin Burton's homebrew setup for connecting multiple cornelius kegs to a single spunding valve. 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). 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 (~30 minutes in). Higher levels of acetic acid are sometimes desirable for Flanders Red Ale style beers.

Mark Trent's demonstration of how easy it is for temperature changes to cause a vacuum and suck-back air into a vessel:

Different airlocks and vessel materials diffuse oxygen at different rates. For example, a set of experiments published by Dr. Enrich L. Gibbs at BetterBottle™ showed that rubber stoppers prevented oxygen transfer more effectively than silicone stoppers, plastic stoppers, and both the "3 piece" airlock and "S" airlock. Solid bungs, however, can build pressure inside the fermenter as the beer slowly ferments, and can pop off due to the pressure (and can cause messes if the vessel becomes pressurized too much). Weekly degassing for a few months while the beer ages is one option with solid bungs. Another option is to rack the beer to a keg and age it in a sealed environment, however, pressure can build up in kegs as well so they should occasionally be partially degassed (some gas should remain in the keg to maintain the seal for corny kegs). See the BetterBottle™ paper for more information. Raj Apte found that HDPE buckets let in far more oxygen than carboy setups, and taking into account the high surface area to volume ratio in homebrew setups versus full-size barrels, oxygen exposure over time on the homebrew level can be a difficult issue to solve. For example, Apte attempted using wooden dowels as stoppers in carboys and found that it let in about the same amount of oxygen as wooden tanks at Rodenbach. However, the swelling of the wooden dowels led some people to crack or destroy glass carboys (therefore this method is not recommended) . If signs of oxygen exposure appear (the growth of a pellicle, the smell of acetic acid or ethyl acetate, etc.), it might be wise to package the beer sooner rather than later, assuming the gravity is stable.

In regards to buckets, MTF members have reported using HDPE buckets successfully for beers aged even 2+ years and soleras. We recommend using a higher quality HDPE bucket with a lid that has a gasket that seals. Avoid plastic-on-plastic lids and screw on lids .

Tank	Volume in Liters	O2 cc/Liter per Year
Burgundy barrel	300	8.5
Rodenbach tank, wood, small	12,000	0.86
Rodenbach tank, wood, large	20,000	0.53
HDPE bucket	20	220
Homebrew barrel	40	23
Glass carboy, 30cm vinyl immersion tube	20	0.31
Glass carboy, silicone stopper	20	17
Glass carboy, wood stopper (not recommended)	20	0.10

Headspace and fermenter size are also concerns when it comes to aging beer with living Brettanomyces. This includes sour beers, non-sour beers with Saccharomyces and Brettanomyces, and 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 CO₂ 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 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 Ideal Gas Law states that unlike solids or liquids of different densities, gasses of different densities eventually mix. See Dr. Chris Colby's explanation of this on Beer and Wine Journal. and this 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 ).

See also:

• Dan Pixley's video on "Airlocks 101 for Sour Beers".
• The oxygen permeability of different plastics.
• Eccentric Beekeeper Sampling Syringe and Purging Wand for tools that help limit oxygen exposure during sampling.
• "Brewing Beer in America Inspired By the Belgian Lambic Tradition" by Ryan Fields, head brewer and blender at Beachwood Blendery.
• Dominick Patrick's HBT thread, "Turning your Fermonster into a complete closed transfer system for cheap!"

Modern Method - Fast Fermentation

Introduction

The short fermentation method refers to an approach for making sour beers that involves successive inoculation of microbes by the brewer to a wort designed for faster attenuation. This approach accomplishes the souring and full attenuation of the wort in a shorter time frame than the traditional method.

In the traditional or long ferment method, the selective availability of carbohydrates to particular microbes allows the activity of those microbes to occur in a natural succession. As the microbes with better competitive ability run out of metabolic resources microbial groups with lower competitive advantage, but wider access to metabolic resources, begin their primary activity. In the short fermentation method, the brewer controls the phases of microbial activity. This allows the brewer to introduce the microbes with the lowest competitive ability to the wort first, allowing them to act on the simplest sugars and establishing their population in the absence of better competing microbes. The order of primary microbial activity in the short fermentation method is, therefore, often the opposite of the order typically observed in the long fermentation method. Further, this approach allows the brewer to maintain temperature profiles that are optimal for each microbial phase. Since the phases are controlled by the brewer, there isn't a need for the longer chain sugars that are generally included in the wort designed for long fermentation because the microbes with lower competitive ability have already been established in the beer by the time the better competing microbes are introduced. For this reason, the beer can fully attenuate within 3-4 weeks of it's final inoculation in many cases. It is important to note however that care should be taken in the decision to bottle these beers in such a short time frame (See bottling section below). Once the beer has reached full attenuation the beer can be packaged, in some cases this can be only 6-8 weeks from brew day (see Mixed Fermentation In Less than 3 Months). A number of biochemical reactions that effect flavor and aroma may still take place over a period of weeks, to months or even years, however, most of these reactions do not involve the production of carbon dioxide so these reactions may take place in the bottle.

Wort Production

The grain bill for a short fermentation sour can be based on nearly any style. In contrast to the low fermentability wort used in the long ferment method, the wort used in the short fermentation method is generally designed to be highly fermentable. This is because the order and timing of microbial inoculation, rather than natural succession of the microbial community, is used to control acidity and fermentation characters. A few modifications to grain bills can be made to increase the fermentability of the wort and accomplish the full attenuation of the wort in a relatively short period of time. These modifications include lowering or removing crystal malts from the recipe and mashing for 90-120 minutes at 149°F/65°C. Extract brewers can steep 2-3 lbs. of crushed, malted 6-row or 2-row base pale malt at 149F in their kettle with their extract in order to increase the fermentability of their wort.

Multi-Stage Fermentation

Conceptual graph of fast souring microbe and media dynamics. Y-axis for each microbe group depicts relative activity which combines in a conceptual sense: growth, acidification of wort, attenuation and production of flavor compounds. Plot drawn by Drew Wham based on concepts discussed in American Sour Beer and Wild Brews .

Matt Miller outlines a "three stage fermentation" process on his blog article Understanding, Brewing, and Blending a Lambic Style Kriek . See the article for a much more detailed process. Matt was also interviewed about his process by James Spencer on the BasicBrewing Radio podcast. In summary, his process is as follows:

1. Produce a low or no hopped wort (see the Standard Method above).
2. After boiling the wort, cool it to 90-120°F (32.3-48.9°C), and run it into the fermenter. The exact temperature depends on the culture being used (see the Lactobacillus page for recommended temperatures).
3. Pitch a pure culture of Lactobacillus, and if possible hold the temperature between 90-120°F (32.3-48.9°C) for 2-4 days (see the Souring in the Primary Fermenter page for more details).
4. After 1-3 days, or after the desired pH is achieved (generally between a pH of 3.0-3.7), cool the wort to 65-70°F (18.3-21.1°C), oxygenate the wort, and pitch a starter of Saccharomyces.
5. After primary fermentation has finished, transfer the beer to a secondary vessel for aging.
6. 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 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 the pitching rate for Brettanomyces in a mixed fermentation probably does not impact flavor. See Brettanomyces secondary fermentation experiment for more details). Also optionally, these additional microbes can be co-pitched along with the Saccharomyces during step 5.
7. Age for 6-18 months, or longer if desired.
8. For the last two months of aging, fruit, spices, and/or oak can be added directly into the fermenter (see Soured Fruit Beer and Soured Herb, Spice, and Vegetable Beer). Also, consider Blending with other sour beers.

Fermentation in Less Than 3 Months

Some brewers have been experimenting with mixed fermentations that can finish within 3 months. This approach to mixed fermentations takes some knowledge of the cultures being used and is considered to be an advanced topic. In general, use cultures that don't produce a lot of off-flavors early on in fermentation. For example, Omega Yeast Lab's Lactobacillus blend (OYL-605) and The Yeast Bay's Lorchristi Brettanomyces blends are good choices. Warning: if mixed fermentation beers are bottled too early, they can result in bottle bombs or gushing bottles. Gareth Young offers his advise to brewers wanting to try mixed fermentations that finish within 3 months :

I typically turn around funky beers, especially roughly saison-ish things, pretty fast. One of the things I like about them is the way they change over time, so I like being able to drink them when they're very young, then drinking them sporadically over the next months as they develop.

Looking through stuff I've posted here, I've found one that I posted about 5 weeks from brew day, and it was good a while before that. It was also dry-hopped and had honey added, which meant it took longer. https://www.facebook.com/groups/MilkTheFunk/permalink/1108334109194802/

Here's some stuff about my process:

1) Lower ABV stuff will be quicker. It doesn't need to be super low, but below 6% is probably wise.

2) Make your wort very fermentable. The more fermentable, the quicker it will hit a stable gravity. I do this by mashing at 63 for two hours, then not sparging too hot, so it's becoming more fermentable all the way to the kettle. Also, don't use any grains with unfermentable (or slow-fermentable) sugars. Just base malt, and maybe some oats/wheat or something.

3) Pitch plenty of yeast. I'd use a culture with lots of healthy Saccharomyces and lots of Brettanomyces, and maybe bacteria, right at the start of primary. This lets Brettanomyces character develop more quickly and helps you hit a stable gravity quicker. I always do this, and there are Brettanomyces aromas coming out of the airlock almost immediately. If you reuse this culture, you'll start selecting for things that get the job done quickly.

4) Pitch your culture low, but then let it rip, and warm it up a bit towards the end of fermentation, if necessary. That should get most of the sugars fermented pretty fast but without the off flavours you can get from pitching hot. Brettanomyces can often clear those up, but it takes time. The less mistakes you leave for your yeast/bacteria to clear up, the sooner it will be ready.

5) Bottle as soon as you hit a stable gravity. If you do it right, your final gravity should be 0.998-1.002 pretty quick. If you bottle soon, I find you don't get any THP (presumably because there's enough healthy Saccharomyces around to ferment the priming sugar without producing it). It was only when I decided I wanted my beer to be a bit clearer, and so started leaving it in primary until it cleared, that I started regularly seeing small amounts of THP developing early in bottle conditioning.

For 4-5%-ish beers, the soonest I've started drinking was 1.5 weeks, but I've started in 2 weeks quite a few times (edit: 4 weeks sometimes). Typically, they hit the final gravity I want within a week, then I bottle them and they're properly carbonated in another week (sometimes less). They obviously develop and get "brettier" and more refined over time, but they're still good, and still funky that quickly.

See also:

• Devin Bell and Dan Pixley presentation at SBC 2017 on quick souring.

Souring Without Brettanomyces

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 ). 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 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 less yeast growth. See the 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, it is claimed that this method allows the yeast character to be expressed more so than with kettle sours. In the case of pitching L. plantarum after fermentation with saison yeast, Bell reported that the beer turned out like a sour saison, where as 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 production from the L. plantarum after primary fermentation. Devin clarified that his "best success" is pitching S. cerevisiae saison strain with a selection of Brettanomyces for primary fermentation. After 5-7 days of fermentation, he pitches L. plantarum (2 shots of GoodBelly or 1 package of Omega Labs OYL-605 for 5-6 gallons of beer) . 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 . See also this thread by Devin Bell and this thread by (Zach) Caroline Whalen Taggart.

See also:

• Devin Bell and Dan Pixley presentation at SBC 2017 on quick souring, including more details on this method.
• General tips on using probiotics to make sour beer.

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. 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.

Reusing a Sour Yeast Cake

Reusing a sour yeast cake can often provide great results. Brewers have reported success 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 depending on how old the yeast cake is (interestingly, Brettanomyces remains more viable over time if it was co-fermented with S. cerevisiae than if it was fermented by itself; i.e. 100% Brettanomyces beers ). By pitching new wort on an old sour yeast cake, these microbes (particularly the Lactobacillus) have access to the simple sugars in the wort . Using a young yeast cake is also a viable option, and may carry over more surviving Saccharomyces cells as well as more viable cells of the other various microbes. In general, rinsing or washing the yeast cake is not necessary (acid washing can kill the bacteria). The beer itself can also be used as an inoculate and might be more desirable so as to avoid trub. If the beer has sat in a barrel Acetobacter and other unwanted microbes might be more present on the surface of the pellicle, and would remain after racking the beer out of the barrel, so some professional brewers advise using beer as an inoculate for this reason . If the yeast cake is particularly old, perhaps say older than 1 year, or has a very low pH (low 3's), then making a starter with the slurry will help guarantee the viability of the microbes. Such a starter can be treated the same as a mixed culture starter that can be assumed to not have any viable Saccharomyces.

Some brewers will harvest a certain amount of trub from their fermenters (500mL for example) and use only this amount to inoculate a new batch of beer. This will allow the brewer to control the amount of dead trub material that goes into the new beer. Michael Tonsmeire often advises that the brewer also pitches a fresh culture of Saccharomyces .

In general, it is advised to pitch a fresh culture of Saccharomyces to ferment the bulk of the wort sugars. This can be done before adding the sour yeast cake, or at the same time. 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 before pitching a fresh culture of Saccharomyces. After 1-4 days, a fresh culture of Saccharomyces is then 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 decided timing on when to pitch the harvest sour yeast cake will affect the acidity 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. It might also be possible to not pitch any fresh yeast and rely completely on the sour yeast cake to fully attenuate the wort. In this case, it might take 3-10 days for the fermentation to begin because this approach would be relying on the Brettanomyces that is alive in the yeast cake, and the growth phase of Brettanomyces can take a week or so. If relying only on the sour yeast cake to fully attenuate the wort, making a starter for the yeast cake is preferable to ensure that the microbes in the yeast cake are viable. A pellicle might also develope early, depending on what types of microbes are in the yeast cake and if they have a tendency to develop pellicles or not. Oxygenate as normal whenever the Saccharomyces is pitched to ensure a healthy Saccharomyces fermentation (Brettanomyces also benefits from small amounts of oxygen, and oxygen does not greatly effect lactic acid bacteria; see Aeration above).

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 could produce widely different results, and the use of 10-30 IBU's in the wort can be used to inhibit the lactic acid bacteria if they become too strong and produce too much acidity.

Storing a Yeast Cake or Sample

The brewer may find that it isn't possible to re-use a yeast cake or a portion of the sour beer to inoculate a new batch of beer/wort right away. The yeast cake or beer sample should be stored in a glass jar in the fridge at a stable temperature in order to preserve the microbes for as long as possible. After more than 3-6 months of storage (depends on how hardy the culture is), a starter should be made for the yeast cake/beer to make up for viability loss. Viability is less important for Brettanomyces if it is not being used as the primary fermenter since pitching rate in secondary appears not to have a large effect (see Brettanomyces secondary experiment), but viability is usually more important for the bacteria, which will be stressed from the long storage. Adding ~2 grams of chalk per liter of the slurry might help to buffer the pH and preserve the viability of all of the microbes for longer (more data is needed to prove this hypothesis). If the yeast slurry/beer sample is not very old and the brewer thinks that there might be residual sugars that could be fermented by the Brettanomyces, leave the lid of the jar slightly loose in case a slow fermentation happens (this can occur even at refrigeration temperatures). Another option that some brewers do is to keep the culture at room temperature and "feed" the culture a small amount of wort every few weeks . This requires a vessel with an airlock.

See the mixed culture starters section for more information on starters for mixed cultures.

Quality Assurance and Avoiding Cross Contamination

See Quality Assurance.

See Also

Additional Articles on MTF Wiki

• Mixed Cultures
• Alternative Bacteria Sources
• Brettanomyces and Saccharomyces Co-fermentation
• Wort Souring
• Lactobacillus
• Pediococcus
• Brettanomyces
• Saccharomyces
• Spontaneous Fermentation
• Commercial Sour Beer Dregs Inoculation
• Brewing Methods
• Quality Assurance

External Resources

• "Microbial Dynamics in Traditional and Modern Sour Beer Production," a peer reviewed review of the role of microbes in sour beer production with a focus on lactobacilli.
• Brewing Sour Beer at Home. The Mad Fermentationist. Michael Tonsmeire.
• Saison panel at GABF 2015 including mixed-fermentaiton saisons.
• "Fundamentals of Sour Beer Fermentation" on Sour Beer Blog.
• "Exploring the Wider Potential of Hops, Saison, and Brett" with Chad Yakobson from Crooked Stave via the Craft Beer and Brewing YouTube channel.
• "Dialing in Mouthfeel and Bitterness in Brett Saisons" with Chad Yakobson from Crooked Stave via the Craft Beer and Brewing YouTube channel.
• "Grist and Mash Choices for Mixed-Culture Farmhouse Beers" with Chad Yakobson from Crooked Stave via the Craft Beer and Brewing YouTube channel.