Changes

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.<ref name=GeuzeKriek>Jef Van den Steen, [[Books#Geuze & Kriek: The Secret of Lambic Beer|Geuze & Kriek: The Secret of Lambic Beer]], 2012</ref> The spontaneous fermentation process has shown considerable variability even among different barrels of beer from the same brewery,<ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384 | The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref> though all spontaneously fermented beers appear to follow a general sequence of microbes, which can be broken into four distinct stages:<ref name=AWAs>Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507|Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref><ref name=Oevelen77 >D. Van Oevelen, M. Spaepen, P. Timmermans and H. Verachtert, [http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1977.tb03825.x/abstract|MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE], 1977</ref><ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384 | The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref>

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.<ref name=AWAs> Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507 | Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref>

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.<ref name=AWAs> Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507 | Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref> 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.

<ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384 | The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref> In the case of Kloeckera, the growth begins prior to Saccharomyces in the first days of fermentation, but are overgrown by Saccharomyces in the following weeks.

The lactic acid bacteria increase in number until around month 7, achieving concentrations of 10⁴ cells/mL wort, which is thought to coincide with the onset of summer and warmer temperatures.<ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384 | The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref> With this in mind, it is thought that the increase in lactic acid bacteria be delayed or hastened by decreasing or increasing the storage temperatures, respectively.<ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384 | The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref><ref name=AWAs>Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507|Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref>

Each reservoir potentially contributes different organisms to the lambic. Samples taken from a lambic brewery indicate that the air above the [[koelschip]] and in the cellar is the primary source of the bacteria in the lambic, while the barrels host much of the yeast, though some yeasts are found in the air and some bacteria come from the barrels as well. <ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384| The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref>

The rapid reproduction of the microorganisms found in lambic also lends to them the ability to evolve on much shorter timescales than those of macroorganisms. Combined with the flora found in lambic (and other spontaneous fermentations) being largely resident inside each brewery,<ref name=AWAs> Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507 | Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref><ref name=Oevelen77 >D. Van Oevelen, M. Spaepen, P. Timmermans and H. Verachtert, [http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1977.tb03825.x/abstract|MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE], 1977</ref> it is reasonable to assume that even the slight geographic separation found between the facilities within the Pajottenland will lead to markedly different strains of bacteria and yeasts responsible for lambic fermentation after several years of operation. Further, brettanomyces isolated from different sources from the same location have shown significant genetic variability<ref name=Crauwels1> S. Crawels et. al. [http://link.springer.com/article/10.1007%2Fs00253-015-6769-9| Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains], 2015</ref> <ref name=Borneman1> A. Borneman et. al. [http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004161| Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates], 2014</ref>, indicating that slightly different handling methodologies and even the surrounding architecture may have an effect on the final product. Thus, it is reasonable to assume that at least part of the distinct flavors found in the lambics of each brewery are due to divergent evolution in their respective microbiomes,<ref name = Witrick1> K. A. T. Witrick [https://vtechworks.lib.vt.edu/handle/10919/19203| Characterization of aroma and flavor compounds present in lambic (gueuze) beer], 2012 </ref> though little literature currently exists in this area. For other breweries, "house strains" developed by propagating favorite yeasts often over a period of years and even decades is commonplace, and often accounts for much of a brewery's specific taste.

Conversely, it has been found that spontaneous fermentations in similar worts in other parts of the world follow similar overall trends.<ref name=AWAs> Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507 | Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref> Yeasts such as Saccharomyces and Brettanomyces and bacteria such as the enterobacteria and Pediococcus are cosmopolitan throughout the world, and all are similar enough to be classified into the same genus by both phenotype and genotype. However, the large degree of geographic separation coupled with the rapid evolutionary rates of these organisms will still lead to measurable changes in both. This is highlighted in the fact that while many lambic-like beers have been brewed in other parts of the world, and are often very similar to lambic itself, they still possess noticeably different sensory characteristics. This can be likened to terroir in wine, as the local microbiome in one location will produce a similar, but not identical, product to that in another location.

Little research exists correlating the season of brewing to changes in the microbiology and chemistry of lambic; however, a delay in the appearance of the late-fermentation bacterial flora in lambic was observed when fermentation started earlier in the brewing season, leading to cooler fermentation temperatures.<ref name=Spitaels > F. Spitaels, A. D. Wieme, M. Janssens, M. Aerts, H.-M. Daniel, A. Van Landschoot, L. De Vuyst, P. Vandamme [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384| The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer], 2000</ref> The flora were indistinguishible after 18 months.

Similarly, a study on spontaneously fermented ales in the United States revealed marked differences between ales brewed in the spring versus those in the winter.<ref name=AWAs>Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507| Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref> The flora broadly follow the same pattern of succession regardless of the season of innoculation, although genetic analysis showed distinct differences between the flora responsible for fermentation occurring in the spring versus the winter. The differences between the organisms found in the wort innoculated at different seasons were largest in the early stages of fermentation and by 36 weeks, there was no longer a significant difference in the flora of in either season's wort.

Spontaneously fermented beers from the United States form a group of beers referred to as American Coolship Ales (or ACAs), American Wild Ales (or AWAs), among other names.<ref name=AWAs>Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507|Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref> Spontaneously fermented beers from other parts of the world are occasionally referred to as "American Wild Ales" as well, though it is unclear if there is any reason to associate any arbitrary spontaneous or even mixed fermentation beer that doesn't fall into another existing style with the United States. At present, there are few satisfactory naming conventions for these beers.

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 yield notably different results after fermentation, even if the overall experience of the two styles of beer is similar.<ref name=AWAs>Nicholas A. Bokulich, Charles W. Bamforth, David A. Mills. [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035507|Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale], PLoS One, 7(4), 2012</ref> <ref name=sour> J. Edwards and A. DiCaprio. [http://www.process-nmr.com/pdfs/Edwards%20-%20SMASH%202014%20-%20MNova%20Users%20Meeting%20-%209-7-14.pdf| When Beer Goes Sour: An NMR Investigation], Mestrelab MNova Users Meeting, SMASH – Atlanta, GA, September 7, 2014</ref> <ref name=Oevelen77 >D. Van Oevelen, M. Spaepen, P. Timmermans and H. Verachtert, [http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1977.tb03825.x/abstract|MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE], 1977</ref>