Microbiology and Biochemistry

The spontaneous fermentation of lambic is a complex process involving a succession of bacteria and yeasts that progresses along with the chemical changes that occur during fermentation [1]. The spontaneous fermentation process has shown considerable variability even among different barrels of beer from the same brewery [2], though all spontaneiously fermented beers appear to follow a general sequence of microbes, which can be broken into four distinct stages [1-3]:

1. An enteric stage, starting around three days after the boil and ending around XX-YY days, in which enteric bacteria dominate. 2. Saccromyces dominance, lasting from three weeks to __, in which Saccromyces cerveciae and other Saccromyces species dominate the beer's yeast flora. 3. Pediococcus dominance, lasting from __ to __, in which Pediococcus and other lactic acid bacteria dominate the bacterial flora. 4. Brettanomyces dominance, lasting from ~ZZ months onward.

A fifth stage of fermentation in the bottle after most brettanomyces fermentation is complete may exist, however research towards the long-term aging of lambic is scant. The various stages of fermentation have a significant degree of overlap with one another as the yeast and bacterial populations live alongside one another, and the times at which they begin and end can vary by up to several months [2].

The microbes present in lambic may come from a variety of sources in the brewhouse and the surrounding environment, including the surrounding air, the walls and ceilings of the brewhouse, and the wooden barrels the beer is kept in [2]. The organisms in lambic are present in different parts of the environment and it is likely that the organisms responsible for lambic come from a number of different places.

Stages of Lambic fermentation

IMAGE FROM PAPER

The enteric stage Lambic wort arrives in the coolship at approximately yy% sugar per weight of water, along with an assortment of proteins and fatty acids and other compounds[ref]. Negligible ethanol is present prior to fermentation, nor is there much of the organic acids that will give lambic its characteristic tartness; the wort has a pH around 4.8, which is similar to the wort of other beers. Enteric bacteria, including ______, _______, and _______ are the first to gain a foothold in this environment, with significant numbers found after three to four days. The enteric bacteria primarily consume glucose, which reduces the gravity of the wort from ___ to ___ after the first three weeks.

Enteric bacteria are responsible for the production of acetic acid, and the pH of the wort falls from around 5 to 4.5 in the first week of fermentation. The 40 to 120 mg/L acetic acid found in the wort after the first week is very close to the amount found in the final product[5,8]. Significant changes to the concentration of acetic acid should not occur until the ethanol has a chance to oxidize in aging in the bottle over many years or even decades [6]. The pellicle that forms on the top of the wort forms around ___ days, and is likely the product of acetobacteria during the enteric phase [9].

Low pH (below ~4.5) and an ethanol concentration higher than ~2% by volume is a hostile environment to the enterobacteria, and Saccromyces species are able to dominate in the wort once these conditions occur around ____ days into fermentation.

Saccromyces dominance After the pH falls below ~4.5 and the alcohol content rises over ~2%, Saccromyces species take over as the dominant organisms in the wort, though Saccromyces is present in large numbers well prior to the dissapearance of the enterobacteria. Saccromyces will remain dominant until at least ___, and usually maintain a considerable presence until ____, when Brettanomyces becomes dominant. Despite Saccromyces' importance to the fermentation, its concentrations remain below 10^7 cells per mL of wort, which is considerably lower than the 10^8 cells/mL found in commercial beers [6].

As in controlled fermentation, Saccromyces is responsible for most ethanol production and attenuation in lambic. The yeasts consume all the major sugars found in lambic wort (glucose, maltose, and even maltotriose). By the end of the Saccromyces phase around __-8 months, the ethanol content of the beer stabilizes at 5-7% by volume and will remain around that value until the end of fermentation[5,7]. Attenuation after Saccromyces fermentation reaches 60-65%, which is known as the "attenuation limit" for conventional beers.

Other bacteria and yeasts also thrive during the Saccromyces phase. ____ and ____ both survive in considerable numbers alongside Saccromyces. MORE SHIT ON OTHER ORGANISMS DURING SACC PHAZE

Pediococcus dominance

-- Lactic acid

The Brettanomyces stage Brettanomyces inherits the role of most prominant yeast genera from Saccromyces around __ months, and continues consuming sugar in the wort. Final attenuation can reach over 80% in lambic through the continued action of Brettanomyces, which is often referred to as "overattenuation" or "superattenuation". This is greater than is usually possible with saccromyces alone, as brettanomyces is able to metabolise sugars that saccromyces cannot.

Refermentation in the bottle Gueuze and other lambic bottled with either some residual sugar left unfermented at the time of bottling or added priming sugars will undergo significant fermentation in the bottle, though all unpasteurized lambic will continue to ferment to some degree there. For lambic bottled after about ____ months without additional sugar, the fermentation in the bottle progresses much as an extension of the Brettanomyces stage of fermentation and negligible carbon dioxide and ethanol production occurrs, leaving most of this lambic still. For lambic bottled younger and those with additional fermentable sugars added at bottling, considerable fermentation by saccromyces occurs in the bottle, causing marked increases in ethanol and the production of carbon dioxide, leading to a carbonated product [ref].

The reactivation of saccromyces fermentation at the addition of additional sugar indicates that the dominance of brettanomyces is brought about by brettanomyces ability to ferment sugars that saccromyces cannot. Once saccromyces has consumed all the available ______ and ______ in the wort, it goes dormant, though does not die completely, and brettanomycs is free to assume the role of primary yeast in the wort. Upon the re-introduction of _____ and _____, the faster-growing saccromyces once again flourishes, until the again the fermentable sugar is consumed and brettanomyces and its other associated yeasts can once again resume the slow procress of the final fermentation.

Continued aging Eventually even the slow-fermenting Brettanomyces runs out of fermentable sugars and fermentation draws to a slow close. The beer will continue to change and evolve over time, though minimal interaction with active yeast occurs. This stage is marked by oxidation and breakdown of the more complex parts of the yeast itself [ref].

While oxidation can occur rapidly due to a break in the fidelity of the seal at the cork, oxidation can still occur without the passage of oxygen through the cork or significant oxygen gas in the headspace due to the transfer of oxygen from an oxygen-containing compound like ____ or ____ in the wort to others. The act of losing an oxygen, or more generally, of losing electrons, is called reduction. Reduction of ____ by ____ to ___ and ___ respectively has been implicated as being of primary importance to the long-term oxidation of flavor compounds in beer [ref], which leads to a characteristic flavor of oxidized beer, commonly described as being of "wet cardboard".

The decomposition of the yeast leads to _____.

Sources of the microbes in Lambic

The microbes found in Lambic may come from a variety of sources, as nearly every surface and even the air found in the brewery are teeming with life. While the air above the coelship is often cited as the source of the microorganisms in Lambic, other sources are now known to play a significant role.

While there are many potential places that the wort can aquire its characteristic flora from, some primary reservoirs to consider are:

1. The air over the wort

2. The ceiling and walls of the facility, from which microorganisms attached to dust or condensation from the hot wort may find their way into the beer

3. The surfaces of the barrels, both interior and exterior, as contamination from the exterior may occur during transfer of the wort.

Other spontaneous fermentations

American Coolship Ale Spontaneously fermented beers from the United States (and occasionally other parts of the world [ref]) form a group of beers referred to as American Coolship Ales or ACAs. ACAs vary in their intended similarity to lambic, with some American producers even going so far as to label their beers "lambics", while other ACAs bear little in common with lambic besides spontaneous fermentation. Due to the geographical separation between the United States and Belgium and the large variations in yeast and bacteria genetics between these different populations, even an ACA wort carefully prepared to be very similar to that of lambic will yeild notably different results after fermentation, even if the overall experience of the two styles of beer is similar [2,3,8].

[1] Lambic book

[2] The spont. ferment. of Lambic Beer

[3] AWAs paper

[4] The microb. diveristy of trad. spont. ferm. lambic beer

[5] MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION

[6] Paper on beer aging and oxidation

[7] Characterization of aroma and flavor compounds present in lambic (gueuze) beer

[8] When Beer Goes Sour: An NMR Investigation

[9]