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=Spontaneous fermentation=
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://journals.plos.org/plosone/article?id=10.1371/journal.pone.00355077|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 />
# An enteric stage, starting around three days after the boil and ending between 30 and 90 days, in which enteric bacteria dominate.
[[file:Salmonella_micrograph.jpg|thumb|left|Electron micrograph of the enteric bacteria Salmonella (pink rods).]]
Lambic wort arrives in the [[koelschip]] at approximately 5% sugar per weight of water, along with an assortment of proteins and fatty acids and other compounds.<ref name=Erbe>T. Erbe and H. Brückner, [http://www.sciencedirect.com/science/article/pii/S0021967300002557| Chromatographic determination of amino acid enantiomers in beers and raw materials used for their manufacture], 2000</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 5, which is similar to the wort of other beers.<ref name=Oevelen77 /> Enteric bacteria, including Enterobacter hormaechei, E. kobei, Klebsiella pneumoniae, and Escherichia coli, 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 ~1.050 to ~1.040 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.<ref name=Oevelen77 />
<ref name=sour> J. Edwards and A. DiCaprio. [http://www.process-nmr.com/Presentation/Edwards%20-%20SMASH%202014%20-%20MNova%20Users%20Meeting%20-%209-7-14.pdf| When Beer Goes Sour: An NMR Investigation], MestrelabMNova Users Meeting, SMASH – Atlanta, GA, September 7, 2014</ref> 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.<ref name=Vanderhaegen1> B. Vanderhaegen, H. Neven, H. Verachtert, G. Derdelinckx [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCAQFjAA&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FGuy_Derdelinckx%2Fpublication%2F222839054_The_chemistry_of_beer_aging__a_critical_review%2Flinks%2F0c960523339c4b25a6000000.pdf&ei=Tq3IVKmfFcGyogSs_YLQCA&usg=AFQjCNFaBrvqDGjqEV2I9uQ73dYh_ParXg&sig2=Z8dY4iDHozbT1eb9JeAdrw&bvm=bv.84607526,d.cGU| The chemistry of beer aging – a critical review], 2006</ref><ref name = Werner> Werner Van Obberghen, '''2. Het algemene productieproces van bier'''</ref> The pellicle that forms on the top of the wort may be the product of acetobacteria during the enteric phase,[9] though most other sources inidcate that the pellicle is the result of Brettanomyces (with Pichia and Candida).<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref>
Low pH (below ~4.5) and an ethanol concentration higher than ~2% by volume is a hostile environment to the enterobacteria, and Saccharomyces species are able to dominate the flora in the wort once these conditions occur around 30 to 60 days into fermentation.
After the pH falls below ~4.5 and the alcohol content rises over ~2%, [[Saccharomyces| Saccharomyces species]] take over as the dominant organisms in the wort, though Saccharomyces is present in large numbers well prior to the disappearance of the enterobacteria. Saccharomyces will remain dominant until at least 6 to 8 months into fermentation, and will maintain a presence, though no longer active, throughout fermentation. Despite Saccharomyces' importance to the fermentation of lambic, its concentrations remain below 10<sup>7</sup> cells/mL of wort, which is considerably lower than the 10<sup>8</sup> cells/mL found in commercial beers.<ref name=Oevelen77 /><ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref>
As in controlled fermentation, Saccharomyces is responsible for most ethanol production and attenuation in lambic. The yeasts consume all the major sugars found in lambic wort (glucose, maltose, and some maltotriose). By the end of the Saccharomyces phase around 8 months, the ethanol content of the beer stabilizes at 5 to 7% by volume and will remain around that value until the end of fermentation.<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref><ref name=Oevelen77 /> Attenuation after Saccharomyces fermentation reaches 60 to 65%, which is known as the "attenuation limit" for conventional beers. Despite being responsible for most of the ethanol in lambic, yeasts of the Saccharomyces genus are not responsible for most of the aroma and flavor compounds that give lambic its distinct sensory characteristics.<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>
Other bacteria and yeasts also thrive during the Saccharomyces phase. [[Kloeckera]] and [[Debaryomyces]] both survive in considerable numbers alongside Saccharomyces.<ref name=Oevelen77 />
[[file:Brettanomyces_micrograph.jpg|thumb|right|Brettanomyces sp.]]
Brettanomyces inherits the role of most prominent yeast genera from Saccharomyces around 8 months, and continues consuming sugars 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 Saccharomyces alone, as Brettanomyces is able to metabolise sugars that Saccharomyces cannot, generally known as "dextrins". In addition, brettanomyces can metabolise laminarin and pectin, and has a greater affinity for the amino acid proline than Saccharomyces<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>. There is some inter-strain variability on what sugars Brettanomyces can metabolize (arbutin, β-methyl-D-glucoside, et al. are only metabolized by some strains of Brettanomyces.)<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>
Brettanomyces has been implicated in producing most of the aroma compounds in Lambic.<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref> Sensory-significant quantities of ethyl acetate and ethyl lactate form at this time from ethanol entering into an ester bond with [[Acetic acid|acetic]] and [[lactic acid]], respectively. In addition, ethylphenols formed from hydroxycinammic acid -- found in the grain used to make the wort -- contribute an odor often described as "horse sweat", "barnyard", or "leather" <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=Lentz1> M. Lentz and C. Harris. [https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0ahUKEwirpvaJlerMAhVBz2MKHXxYB_kQFggnMAE&url=http%3A%2F%2Fwww.mdpi.com%2F2304-8158%2F4%2F4%2F581%2Fpdf&usg=AFQjCNHZB4IHgQasVxVL3JjdmMcWjdFIUw&sig2=pJ8f-mmJKAYHIfO5xj7GhQ| Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast], 2015</ref>. The esterization process is greatly helped by the enzyme esterase provided by Brettanomyces. However, the enzymatic esterization is highly reversible and esters found in high concentrations in the lambic prior to the presence of the esterase will often achieve a lower equilibrium at the end of fermentation. This is the case with iso-amyl acetate, which is produced by Saccharomyces and is a characteristic odor compound in many other beers.
Tetrahydropyridines (THPs) produced by Brettanomyces (as well as some Lactobacilli) have a wide variety of odors and give lambic much of its "mousey" aroma, as well as cider- and horse-like aromas, though the concentrations and thus smells of THPs are variable.<ref name=Heresztyn1> T. Heresztyn [http://ajevonline.org/content/37/2/127.short| Formation of Substituted Tetrahydropyridines by Species of Brettanomyces and Lactobacillus Isolated from Mousy Wines], 1986</ref> Other important odor and flavor compounds produced by Brettanomyces include 4-ethylphenol, 4-ethylguaiacol, and isovaleric acid. 4-ethylphenol produces barnyard and horsey flavors which can taste like Band-aids in higher concentrations. 4-ethylguaiacol lends spicier flavors of bacon and cloves and can be smoky, while isovaleric acid gives lambic its sweaty and cheesy flavors and odors.
Around 16 months after the start of fermentation, during this stage, the pH of the beer reaches a minimum of about 3.0, which then rises slightly in the following months to ~3.2 to 3.4,<ref name = EtF> [http://embracethefunk.com/ph-readings-of-commercial-beers/| Embrace the Funk's list of beer pH]</ref><ref name=Oevelen77 /><ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref> perhaps due to the enzymatic esterification of organic acids by Brettanomyces.
=Refermentation in the bottle=
The ongoing process of lambic aging after its maturation is a very complex process and has both purely chemical as well as biochemical aspects. Much of the present literature is general to all beer.
Eventually even the slow-fermenting Brettanomyces runs out of fermentable sugars and fermentation draws to a prolonged 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 name=Dalgliesh >C. E. Dalgliesh, Flavour stability, [http://www.europeanbreweryconvention.org/EBCmain/organisation/publication.php | Proceedings of the European Brewery Convention Congress], 1977</ref> <ref name=Vanderhaegen >B. Vanderhaegen, H. Neven, H. Verachtert, and G. Derdelinckx, [http://www.sciencedirect.com/science/article/pii/S0308814605000865|The chemistry of beer aging – a critical review], 2006</ref>
The decomposition of the yeast progresses primarily through the action of their own enzymes in a process called autolysis. Autolysis releases a large number of other enzymes, which has the secondary effect of breaking down many other components of the beer.<ref name=Vanderhaegen1> B. Vanderhaegen, H. Neven, H. Verachtert, G. Derdelinckx [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCAQFjAA&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FGuy_Derdelinckx%2Fpublication%2F222839054_The_chemistry_of_beer_aging__a_critical_review%2Flinks%2F0c960523339c4b25a6000000.pdf&ei=Tq3IVKmfFcGyogSs_YLQCA&usg=AFQjCNFaBrvqDGjqEV2I9uQ73dYh_ParXg&sig2=Z8dY4iDHozbT1eb9JeAdrw&bvm=bv.84607526,d.cGU| The chemistry of beer aging – a critical review], 2006</ref> The release of esterase leads to the increased destruction of iso-amyl acetate and other esters, causing a loss of its light, fruity odor. Not all esters are broken down by this process, and a number of esters are formed during aging. These include ethyl 3-methyl-butyrate and ethyl 2-methyl-butyrate which contribute a light floral or even tropical fruit odor.<ref name=Bohmann2> J. J. Bohmann [http://www.brewingscience.de/index.php?tpl=table_of_contents&year=1985&edition=0004&article=56947| Zum Alterungsverhalten des Bieres. 4 Teil, Kombinierte Alterungsversuche durch Begasung mit Kohlendioxid, Stickstoff, Luft und Sauerstoff], 1985</ref><ref name=Bohmann1> J. J. Bohmann [http://www.brewingscience.de/index.php?tpl=table_of_contents&year=1985&edition=0003&article=56946| Zum Alterungsverhalten des Bieres. 3. Teil, Der Einfluß der Strahlungsbelastung, dargestellt am Beispiel 2-Methyl-2-buten und Isopren], 1985</ref> The release of proteases by autolysis causes a breakdown of long protein chains, with its concordant thinning of mouthfeel and reduction in head. While autolysis is often seen as undesirable, it can contribute to a pleasant mouthfeel as well as some of the characteristic flavors found in very old lambic.<ref name="Oxford pg 54">J. Robinson (ed) ''"The Oxford Companion to Wine"'' Third Edition pg 54 Oxford University Press 2006 ISBN 0-19-860990-6</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 oxidizing compound in the wort to others. The act of losing an oxygen, or more generally, of losing electrons, is called reduction.
Production of (E)-2-nonenal (as well as other linear aldehydes) has been implicated as being of primary importance to the long-term oxidation of flavor compounds in beer,<ref name=Jamieson > A. M. Jamieson, E. C. Chen, and J. E. A. Van Gheluwe, A study of the cardboard flavour in beer by gas chromatography, [http://www.asbcnet.org/publications/journal/Pages/default.aspx | Proceedings of the American Society of Brewing Chemists], 1969</ref> which leads to a characteristic flavor of oxidized beer, commonly described as being of "wet cardboard". However, many other processes and compounds are important to the aging of beer as well.
The Maillard reaction, also responsible for the browning of toast and steak among many other things, occurs in the unfermented reducing sugars left in the beer during extended aging, leading to a darker brown color as well as a slight burnt toast flavor.<ref name=Vanderhaegen1> B. Vanderhaegen, H. Neven, H. Verachtert, G. Derdelinckx [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCAQFjAA&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FGuy_Derdelinckx%2Fpublication%2F222839054_The_chemistry_of_beer_aging__a_critical_review%2Flinks%2F0c960523339c4b25a6000000.pdf&ei=Tq3IVKmfFcGyogSs_YLQCA&usg=AFQjCNFaBrvqDGjqEV2I9uQ73dYh_ParXg&sig2=Z8dY4iDHozbT1eb9JeAdrw&bvm=bv.84607526,d.cGU| The chemistry of beer aging – a critical review], 2006</ref>
Many other reactions occur as lambic ages that are not elaborated upon here in the interests of brevity.
==Geographical variation==
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 /><ref name=Oevelen77 /> 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 /> 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.
As an example, Brettanomyces shows significant genomic variation between strains, as well as a corresponding variability in their metabolisms, indicating that different strains of Brettanomyces bruxellensis will lend markedly different sensory characteristics to lambic.<ref name = Conterno1> L. Conterno, C. M. L. Joseph, T. J. Arvik, T. Henick-Kling, and L. F. Bisson [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0CC0QFjAB&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FC_M_Joseph%2Fpublication%2F236843438_Genetic_and_Physiological_Characterization_of_Brettanomyces_bruxellensis_Strains_Isolated_from_Wines%2Flinks%2F0c960528eb2e4a0513000000.pdf&ei=t6HKVJbHJZWyoQT2qoCYBg&usg=AFQjCNFMfoISmHrGfWtGfDHJWJI4w25vOw&sig2=u9Loxwy3zVWXK4gy8vn_2g&bvm=bv.84607526,d.cGU| Genetic and Physiological Characterization of Brettanomyces bruxellensis Strains Isolated from Wines], 2006</ref><ref name=Borneman1 /><ref name = Yakobson> [http://www.brettanomycesproject.com/dissertation/ Brettanomyces Project Dissertation], Chad Michael Yakobson</ref><ref name = Curtin> C. D. Curtin, J. R. Bellon, P. A. Henschke, P. W. Godden, and M. A. de Barros Lopes [http://www.pubfacts.com/detail/17233769/Genetic-diversity-of-Dekkera-bruxellensis-yeasts-isolated-from-Australian-wineries.| Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries], 2007</ref><ref name = Agnolucci> M. Agnolucci, I. Vigentini, G. Capurso, A. Merico, A. Tirelli, C. Compagno, R. Foschino, M. Nuti [http://www.researchgate.net/publication/222660112_Genetic_diversity_and_physiological_traits_of_Brettanomyces_bruxellensis_strains_isolated_from_Tuscan_Sangiovese_wines| Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines], 2009</ref>
==Seasonal variation==
==Spontaneous fermentation in other traditional drinks and foods==
Other spontaneous fermentations exist as well, such as Chicha, a corn-based lightly alcoholic drink indigenous to the Andes. Much like the spontaneous fermentation of lambic, Chicha possesses its own set of yeasts, dominated by strains of Saccharomyces responsible for its unique characteristics.<ref name=Vallejoa >J. A. Vallejoa, P. Mirandaa, J. D. Flores-Félixb, F. Sánchez-Juanesc, J. M. Ageitosa, J. M. González-Buitragoc, E. Velázquezb, T. G. Villaa, [http://www.sciencedirect.com/science/article/pii/S0723202013001513| Atypical yeasts identified as Saccharomyces cerevisiae by MALDI-TOF MS and gene sequencing are the main responsible of fermentation of chicha, a traditional beverage from Peru], 2013</ref>
Brettanomyces and other wild organisms, despite often being spoilage organisms in wine, are responsible for some major sensory characteristics of some wine styles.<ref name=Tristezza> M. Tristezza, C. Vetrano, G. Bleve, G. Spano, V. Capozzi, A. Logrieco, G. Mita, F. Grieco [http://www.sciencedirect.com/science/article/pii/S0740002013001408 | Biodiversity and safety aspects of yeast strains characterized from vineyards and spontaneous fermentations in the Apulia Region, Italy], 2013</ref><ref name=Medina> K. Medina, E. Boido, L. Fariña, O. Gioia, M.E. Gomez, M. Barquet, C. Gaggero, E. Dellacassa, F. Carrau [http://www.sciencedirect.com/science/article/pii/S0308814613005025 | Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae], 2013</ref> Other minor yeasts found in lambic, such as Pichia and Kloeckera, have also been found in unspoiled wine.<ref name=Tristezza> M. Tristezza, C. Vetrano, G. Bleve, G. Spano, V. Capozzi, A. Logrieco, G. Mita, F. Grieco [http://www.sciencedirect.com/science/article/pii/S0740002013001408 | Biodiversity and safety aspects of yeast strains characterized from vineyards and spontaneous fermentations in the Apulia Region, Italy], 2013</ref><ref name=Medina> K. Medina, E. Boido, L. Fariña, O. Gioia, M.E. Gomez, M. Barquet, C. Gaggero, E. Dellacassa, F. Carrau [http://www.sciencedirect.com/science/article/pii/S0308814613005025 | Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae], 2013</ref>