Culturing Lambic Organisms


To brew lambic-style beer it is advantageous to know the basics of yeast culturing. It is a further help to know how to culture the various organisms specifically involved in lambic fermentation. These include species of Saccharomyces and Brettanomyces yeast and Pediococcus bacteria. This document will concentrate on the culturing of the latter two genera.

Any type of Saccharomyces can be used for the primary fermentation of a lambic-style beer since it will contribute little if anything to the overall flavor profile of the finished product. One can find any number of articles and books on how to culture these yeast and maintain them in the home brewery. (Ref: Leistad? Rajotte?) Also check out the yeast FAQ for basic culturing tips.

It is the other organisms involved which are more difficult to culture and maintain without some experience and knowledge of how they grow. The yeasts Brettanomyces bruxellensis and B. lambicus and the bacteria Pediococcus spp. are fastidious organisms that require very specialized nutrients and environmental conditions to thrive and remain viable. Unless you are going to brew lambic-style beers more than once or twice a year you may be better off buying new cultures each time rather than trying to maintain them in a home environment. If you do feel the need to maintain the cultures at home then you should become very familiar with the culturing of normal brewing yeast first.

Both Brettanomyces and Pediococcus produce acid as a normal metabolic by-product therefore they need to be maintained on a medium which can help neutralize it. Otherwise the acid will lead to a large drop in pH and the organisms will die. The most readily available food grade acid-neutralizing agent is calcium carbonate, also called precipitated chalk. A concentration of 2% in the growth media or agar will provide adequate buffering capacity. Calcium carbonate (CaCO3) is essentially insoluble though and this can present problems if it is used in making wort agar. To prevent the CaCO3 from sinking to the bottom of the agar one should cool the wort agar solution to 45-50°C, with constant swirling, prior to pouring it into the tubes or dishes. When it is used in liquid media one can gently swirl the media with the culture growing in it once a day to help prevent any stratification of the acid in the solution and aid the neutralization of it with the CaCO3. As the lambic organisms grow in the liquid media the acid produced will react with the CaCO3 causing it to be solubilized over time. Even when a culture reaches maximum confluence there may still be some CaCO3 unneutralized, but this is not anything to worry about as the acid produced by the growth in the wort will dissolve it in time. I would like to discuss in more detail the individual organisms and how to maintain them. The Brettanomyces yeast can be grown on wort agar which incorporates 2% CaCO3 as noted previously. The wort itself should be of a gravity of approximately 1.040 and preferably made from an all grain wort due to the possible lack of nutrients malt extract (refs). Due to the fastidious nature and acid production by Brettanomyces they need to be transferred to new slants more often than do normal saccharomyces. I recommend they be transferred to new slants at least every 2 months. Longer intervals may lead to cultures which are no longer viable.

Because of this frequent manipulation there is a greater chance the yeasts themselves may become contaminated with other yeast, mold or bacteria. Thus your technique needs to be very good and you need to pay attention to details such as proper sterile culture techniques. If your culture does become contaminated with another yeast you can prepare a dilute suspension of the yeast and streak it out on a petri dish which has wort agar with 2% CaCO3 and also incorporates 10 ug/ml cycloheximide (make up stock solutions of cycloheximide in ethanol at up to 50 mg/mL). (Caution: cycloheximide is a toxin (LD50 150 mg/kg i.v. in mice), a mutagen, a teratogen. . .)

Is this info correct? Dilute alkali will inactivate cycloheximide.

As the colonies grow watch for a zone of clearing around the individual colonies. Agar prepared with calcium carbonate will be opaque and white in color and as the Brettanomyces grow the acid they produce will dissolve the calcium carbonate and create the zone of clearing. Generally only non- saccharomyces yeast will grow on media with cycloheximide in it and those that do grow and have zones of clearing around them are likely to be Brettanomyces. You can then pick the colonies off and re-culture them on cycloheximide-free media. This is by no means a definite technique for the isolation and characterization of Brettanomyces. If you have doubts at all then you may be better off buying new cultures and/or locating a microbiologist who will work for homebrew. The only absolute way to characterize a culture is through extensive fermentation, assimilation and morphological testing, don't believe it if someone tells you otherwise. Pediococcus Pediococcus is more difficult to grow and maintain than is Brettanomyces due to its more complex nutritional and environmental requirements. Pediococcus grows best in liquid, rather than solid media and this increases the chances for unseen contamination. Unless you have access to a 1000 X power microscope and have culturing experience I recommend that you do not try to maintain this bacteria at home for any length of time. MRS is the preferred medium for growing and maintaining Pediococcus. It is rather expensive but provides the necessary nutrients and buffers for optimal growth over extended periods. It has been my experience as well as that of others that bacteria can loose their hop resistance if grown in an unhopped medium for extended periods. Thus if you are going to try to keep cultures going over a period of time I suggest you had iso-alpha acids to the medium using some type of hop extract. Use of real hops leads to particulate matter which causes the medium to become cloudy and less than ideal for visual monitoring of growth. A suggested level is in the 15-25 IBU range.

Passage Interval

Starters With regards to preparing starters for either organisms a wort of specific gravity 1.040 with 2% CaCO3 will work just fine. You may also want to add some hops, again to achieve a range of 15-25 IBU. You should also follow a scale up procedure for both organisms so as to reduce the risk of other organisms over growing the cultures. Such procedures have been outlined elsewhere. Be aware that Brettanomyces is a very slow grower as compared to other yeast and generally does not develop a large kraeusen head either. The starter should develop a typical Brettanomyces aroma due to the various fermentation by products. You should notice a distinct acid aroma along with aromas often described as horsy or mousy. Also the starter will develop an acidic, mousy, earth taste if the yeast is indeed Brettanomyces. As the yeast grow the acid they produce will cause the CaCO3 to dissolve and the fine white precipitate from it will become less noticeable with time. Due to the additional vitamin requirements of Pediococcus you may want to add 10% apple or tomato juice to the starter. Another alternative is to use 0.5% dried brewers yeast. Pure yeast extract is even better but is expensive and not absolutely necessary. I prefer to clarify my wort by boiling it and then chilling it close to freezing and pouring off the clear liquid and canning it. This way I can monitor the growth of the bacteria more easily. As the bacteria reaches confluence a clear zone will develop at the top of the liquid and the rest of the liquid will be very cloudy due to the large amount of bacteria. This brings me to a discussion of starter volume to use when brewing lambic style beers. For most other beers you want to pitch a large volume of yeast to ensure a rapid fermentation and decrease the risk of contamination by wild yeast and bacteria. Well lambic style beers require you to throw caution to the wind. I typically use starters of no more than 50 ml of normal ale yeast and Brettanomyces. And with Pediococcus I will typically use a volume of no more than 25 ml. If the yeast and bacteria are healthy there is no reason they should not grow in the wort using these volumes. Remember this beer will ferment of a year or more and the bacteria and yeast have plenty of time to grow. Also as the acid producing strains grow they will kill off any other organism due to the lower than normal pH, increase in alcohol and reduction in sugars that are fermentable by other organisms. But if you feel the need to use larger starters to put yourself at ease then by all means go ahead. Just remember the beer will still take a year or so to develop the proper character.

Inoculation Schedules

Traditionally after the wort is boiled it is allowed to cool overnight and during this time it becomes infected with various bacteria and yeast. these come from the air and the surrounding brewery environment and cause the wort to ferment. I know of a few individuals here in the US who have followed this approach with mixed results. More often than not the resulting product is not a very close approximation of what real a lambic is like. (define lambic, gueuze etc.)

The majority of lambic style brewers I have corresponded with prefer to use pure cultures to inoculate their wort and ferment it. This gives them a bit of control over the process which is can still lead to a largely unpredictable result.

In spontaneously fermented lambic there is a succession of growth of the various organisms as described by Guinard. With pure culture lambic one has numerous options regarding when and how to add the various microorganism. These range from simply adding all the cultures right after cooling to adding each organism separately at various times to mimic the natural process.

The whole process is further complicated by the decision of what size inoculum to use. Should one try to mimic the natural conditions where the initial number of cells/ml wort is very small or should one use larger cell numbers to ensure proper growth of these fastidious organisms There is not a simple answer to these questions and again many techniques have been and need to be tried.

Brettanomyces and Pediococcus are slow-growing microorganisms requiring special environmental conditions to grow. Even under the best conditions traditional lambic brewers have problems with some cask not fermenting properly because the various organisms fail to thrive. The same thing can happen to those trying to make lambic style beers using pure cultures. If the environmental conditions are not correct to start with or change to quickly one or more of the microorganisms may fail to grow or grow to much and the resulting beer will lack the proper balance of flavors. I have tasted a number of lambic style beers where this is the case. Of course I also do not have any definite answers as to how to ensure the balanced growth of all the organisms either.

The starters I have used have ranged in size from 10 to 100ml for the various yeast and bacteria I have used. I have also used a variety of inoculation schedules for these various organisms. At this point in time many of these beers are still fermenting and it will be at least another 6 months to a year before I get any final results. So at this point in time I am not sure what starter size and additions schedule will work the best. The one thing I do know is that you need to have a healthy cultures to start with a provide them with the proper environment with all the necessary nutrients they need to grow.

You should also check out Todd Gierman's Anarchist's pLambic, for an easier, more radical approach.

Don't Rack!

After you settle on an inoculation schedule and size there are things you can do to achieve good growth of the various yeast and bacteria involved. First of all you should have a wort (wort and buffering?) high in dextrins and unconverted starch. This can be achieved by using 30-40% raw wheat in the grist and various mashing techniques as already described. Once you have the cooled wort and it is inoculated you should allow it to remain in one vessel for the entire fermentation process. This runs counter to the typical practice of racking the beer to a new container after primary fermentation is complete. By not racking one does not remove the nutrient-rich trub and yeast which will undergo autolysis and provide nutrients for the Brettanomyces yeast and Pediococcus bacteria. By not racking you ensure that enough of the proper nutrients are present.

Temperature control is not as crucial in lambic brewing as with other styles of brewing, but one should still try to avoid extreme temperatures and fluctuations. A temperature that is too high or low may lead to little or too much growth of one or more of the various microorganisms. In Belgium the temperature of the lambic cask rarely exceeds 25°C. At the same time don't get too concerned about the temperature since it may be hard to find an ideal temperature environment for the 1-2 years it will be fermenting.

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