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===Microbial Succession During Fermentation===
====First Stage: Enterobacteria====
The number of different species found in lambic and spontaneously fermented beers is very large and diverse from brewery to brewery and batch to batch, however, scientific research in Belgium and the US has shown a regular general pattern to the microbial succession of spontaneous fermentation beer at the genus level, with only minor genera differences between Belgian lambic beers and American spontaneous ale. This difference is attributed to different microbes being present in different breweries <ref name="Van Oevelen et al., 1977">[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. Van Oevelen et al., 1977.]</ref><ref name="Bokulic et al., 2012" /><ref name="Spitaels et al., 2014">[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095384#pone-0095384-g004/ The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer. Spitaels et al., 2014. DOI: https://doi.org/10.1371/journal.pone.0095384.]</ref><ref name="Spitaels et al., 2015" /><ref name="Roos_2018_2" /><ref>[http://www2.parc.com/emdl/members/apte/slides_nchf.pdf Raj Apte Concepts of sour Beer, 2004]</ref>. The first stage, which lasts for approximately 1 month <ref name="Van Oevelen et al., 1977" /><ref name="Martens et al., 1992">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1992.tb01126.x/abstract/ Martens et al., 1992]</ref>, is dominated by [https://en.wikipedia.org/wiki/Enterobacteriaceae enterobacteria] and [http://laboratoryresearch.blogspot.com/2008/07/yeasts-and-yeastlike-fungi.html?m=1 oxidative yeasts] that produce large amounts of DMS which can be smelled during the early stages of fermentation (see [[Dimethyl Sulfide]] for more details). Although spontaneous ales have a common pattern of fermentation by groups of genera of microbes, the diversity in specific species is large across different lambic producers and American spontaneous ale producers (although data for American spontaneous ale producers is limited). In American spontaneous ale producers, ''Klebsiella'' spp., ''Enterobacter'' spp.,'' Pectobacterium carotovorum'', and ''Serratia ureilytica'' have been found. In Belgian lambic producers, ''Enterobacter'' spp., such as ''Enterobacter aerogenes'', ''Enterobacter cloacae'', ''Enterobacter hormaechei'' and ''Enterobacter kobei'', ''Klebsiella aerogenes'', ''Klebsiella oxytoca'', ''Klebsiella varicola'', ''Escherichia coli'', ''Hafnia alvei'', ''Hafnia paralvei'', and ''Citrobacter freundii'', have been found in lambic, with ''E. cloacae'' and ''K. aerogenes'' as the most frequently found ones. Although these enterobacteria contribute little in terms of gravity drop over the first month of fermentation (they mostly consume sucrose in the wort), they do contribute aroma and flavor compounds and precursors during the initial stages of spontaneous fermentation, particularly acetoin, 2,3 butanediol, acetic acid, lactic acid, succinic acid, DMS, acetaldehyde, long-chain fatty acids (these play a role in both flavor impact and providing nutrients for yeast later in the fermentation process), and small amounts of glycerol, ethyl acetate, and higher alcohols which might form esters in the later stages of fermentation. Enterobacteria can also contribute to the production of [https://en.wikipedia.org/wiki/Biogenic_amine biogenic amines] in fermented foods and beverages, including spontaneously fermented beers. Enterobacteria usually disappear after 30-40 days of fermentation due to the increase in ethanol, decrease in pH, and a decrease in food availability <ref name="Martens et al., 1992" /><ref name="Roos_2018">[https://www.ncbi.nlm.nih.gov/pubmed/30246252?dopt=Abstract Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production. Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>, although one study by Curtin et al. reported finding at least small populations of enterobacteria as late as up to 4.5 months <ref name="curtain_asbc_2018">[https://www.asbcnet.org/lab/webinars/webinars/Pages/funkyFermentationsWebinar.aspx Chris Curtin. ASBC webinar: "Funky Fermentations". 12/12/2018. Retrieved 01/03/2019.]</ref>(~25 minutes in). Acidifying the wort to a pH below 4.5 before cooling and exposing to ambient microbes in a coolship can partially eliminate the enterobacteria phase of spontaneous fermentation and thus avoid or limit biogenic amine production, which is a practice for some lambic breweries <ref name="Spitaels et al., 2015" /><ref name="Roos_2018_2" />. While enterobacteria and oxidative yeasts are not considered to be a part of the core microbes in spontaneous fermentation, it has been shown that ''Saccharomyces cerevisiae'' is metabolically stimulated when co-fermented with some of these species, allowing the ''S. cerevisiae'' to consume more glucose and nitrogen and to more quickly replicate <ref name="Roos_2018" />. De Roos et al (2018) reported significant populations of the enterobacteria species ''Klebseilla variicola'', ''Klebsiella oxytoca'', and the yeast species ''Hanseniaspora uvarum'', ''Saccharomyces cerevisiae'' during the first week or two of lambic fermentation that was pre-acidified (see [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252343/figure/F3/?report=objectonly Figure 3]). '''Wort or beer fermenting during this stage should not be consumed due to the fact that some of these are pathogenic bacteria and pose potential health risks.''' Oxidative yeasts are also present during the first stage of fermentation, including species of ''Rhodotorula'', ''Candida'', ''Cryptococcus'', ''Hanseniaspora'', and ''Pichia'', some of which might survive pre-acidification <ref name="Bokulic et al., 2012" />. Zach Taggart found that this initial stage also corresponded with a pH drop from 5.0 to 4.5 in 6 days and the aroma went from sweet-smelling wort to phenolic and a light burnt rubber character during this time in one batch of spontaneous fermentation <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2360399550654912/ Zach Taggart (using his wife's Facebook account). Milk The Funk Facebook group post on analysis of spontaneous fermentation at 42 North Brewing Co. 11/09/2018.]</ref>.
The second stage of spontaneous fermentation is dominated by ''Saccharomyces'' species (predominantly ''S. cerevisiae'', ''S. bayanus'', and ''S. pastorianus'', the latter often being present towards the end of this phase). ''Hanseniaspora uvarum'' has also been reported in some but not all lambic fermentations playing a major role in starting the second stage of spontaneous fermentation, which is characterized by ethanol production. Most of the attenuation is accomplished during this stage with the depletion of monosaccharides, disaccharides, and trisaccharides consumed in that order (glucose/fructose is consumed first by the ''S. cerevisiae'', and then maltose/maltotriose are gradually depleted until they are gone by the end of the second stage). Ethanol, methyl-1-butanol, and succinic acid are the main compounds produced during this stage for wort that has been pre-acidified. This stage lasts approximately 3-4 months. One study also found populations of ''Kazachsania'' yeast species and ''Cellulosimicrobium'' yeast species early on in the second stage <ref name="Roos_2018_2" />. In addition to the bulk of the overall ethanol production, this phase also sees the production of higher alcohols and the synthesis of esters, especially isoamyl acetate, as well as glycerol, caprylic acid, and capric acid <ref name="Van Oevelen et al., 1977" /><ref name="Roos_2018" />. It has been reported by some brewers that this stage might begin as early as 3-14 days and corresponds with a drop in pH below that of regular beer, indicating that the first stage for some spontaneous fermentations might be shorter and faster than reported in the other literature <ref>[http://www.spontanmanc.co.uk/?p=66 Zach Taylor of Chorlton Brewing Co. "The Lab Work Begins". Spontanmanc blog. 08/01/2018. Retrieved 08/29/2018.]</ref>. MTF members (both homebrewers and professionals) have observed yeast fermentation activity typically at 3-7 days <ref>[https://www.facebook.com/events/666424196868756/ Various MTF members. Milk the Funk - Collaboration Brew #3: Spontaneous. 05/01/2017. Retrieved 08/29/2018.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1571139996247542/?comment_id=1571597289535146 Raf Soef, James Howat, Levi Funk. Milk The Funk Facebook thread on how long it takes for yeast to start fermenting in a spontaneous fermentation. 2017.]</ref>. However, these reports are anecdotal based on visual fermentation and microbe analysis was not done in many cases. De Roos et al. (2018) reported that for wort that is pre-acidified to a pH of 4.5, and after an initial drop in pH to 3.8 by enterobacterial and acetic acid bacteria, the pH rose to 4.0 during the secondary fermentation phase, indicating that the yeast consumed some of the organic acids that were produced during the initial enterobacteria phase <ref name="Roos_2018_2" />.
The ''[[Saccharomyces]]'' dominated stage of fermentation is followed by prolonged and gradual acid and flavor development accompanied by the final points of attenuation, which lasts anywhere from 2 to 10 months <ref name="Roos_2018" />. In some descriptions this is split into an "acidification phase" which is dominated by lactic acid bacteria (LAB), primarily ''[[Pediococcus]]'' and sometimes ''[[Lactobacillus]]'', and a "maturation phase" driven by ''[[Brettanomyces]]'' <ref name="Van Oevelen et al., 1977" />. Other sources describe these as one extended maturation phase with acidification from ''Pediococcus'' and ''Brettanomyces'' growth occurring simultaneously <ref name="Spitaels et al., 2015" /><ref name="Bokulic et al., 2012" /><ref name="Spitaels et al., 2014" />. When the wort is pre-acidified, the acidification and maturation phases overlap <ref name="Roos_2018" />. Other yeasts such as ''Candida'', ''Cryptococcus'', and ''Torulopsis'' species have also been isolated from mature lambic, although their impact other than possibly being involved in the formation of a pellicle is unknown <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.1977.tb03825.x MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE. D. Van Oevelen M. Spaepen P. Timmermans H. Verachtert. 1977. DOI: https://doi.org/10.1002/j.2050-0416.1977.tb03825.x.]</ref>. As many of the flavor and aroma characteristics that we associate with spontaneously fermented beer are produced during this slow maturation/acidification phase, allowing sufficient aging time is important when producing spontaneously fermented beers <ref name="Van Oevelen et al., 1976" /><ref name="Spaepen et al., 1978" />. Homebrewer Caleb Buck reported data on several batches of homebrewed spontaneously fermented beer and observed a slower drop in gravity for some batches than others over about a 7 month period (see [http://www.archaicpursuit.com/2018/08/2017-coolship-experiment-hopping-rate.html?m=1 this graph for details]). De Roos et al. (2018) reported a gradual increase in glucose, maltose, and maltotriose from week 7 to month 6 due to the degradation of maltooligosaccharides (higher chain sugars) <ref name="Roos_2018_2" />.
====Third Stage: Acidification====
The acidification phase is also accompanied by the growth of acetic acid bacteria (AAB), which can be undesirable if this growth is excessive since it leads to greater [[Acetic Acid|acetic acid]] production (in high quantities, acetic acid smells and tastes like vinegar and is very harsh on the palate and throat) as well as acetoin. These microbes include species from the genera of ''Acetobacter'' and ''Gluconobacter''. These microbes are dependent on oxygen in order to metabolize ethanol into acetic acid (with acetaldehyde produced as an intermediary step) and acetoin from lactic acid and are found on the surface of the wort where oxygen is available. The beer/air interface (or surface of the beer that interfaces with the air above it) is also where higher concentrations of acetic acid and acetoin are found due to the AAB being present there rather than deeper within the beer (this is similar to [[Flanders Red Ale]]). It has been shown that the species of AAB found in lambic and American spontaneous ales have adapted to high concentrations of ethanol and acetic acid <ref name="Roos_2018" />. With the flavor threshold of acetic acid in beer being 90 ppm <ref>[https://www.aroxa.com/beer/beer-flavour-standard/acetic-acid Aroxa website. "Acetic Acid". Retrieved 11/19/2018.]</ref>, and the levels of acetic acid in Belgian gueuze/lambic being reported in the range of 727-2240 ppm, acetic acid levels in this range is an important flavor compound in spontaneously fermented beers <ref>[http://beachwoodbbq.com/pdf/BBAIBLTBLENDERY.pdf Ryan Fields. "Brewing Beer in America Inspired By the Belgian Lambic Tradition". 2018.]</ref><ref name="Spitaels et al., 2015" />. De Roos et al. (2018) reported high numbers of ''Acetobacter pasteurianus'' from month 3 to month 6, with it disappearing around month 9-13 as ''Pediococcus damnosus'' took its place. They also reported finding significant levels of ''Acetobacter orientalis'' during week 2 and 3 of lambic that was pre-acidified <ref name="Roos_2018_2" />. Curtin et al. (2018) showed that acetic acid bacteria came and went at various random points within a 0-4.5 month period of fermentation <ref name="curtain_asbc_2018" />(~26 minutes in).
====Fourth Stage: Maturation====
The fourth and last phase of spontaneous fermentation, also known as the extended maturation phase, is dominated by ''[[Brettanomyces]]'' yeast, which is a genus of yeasts that are highly tolerant of low pH, high alcohol, and can survive in low-nutrient conditions, as well as lactic acid bacteria from the genera ''[[Pediococcus]]'' and to a lesser extent ''[[Lactobacillus]]'' and ''Pichia'' yeast species. This phase generally begins somewhere around month four to eight, with these microbes completely dominating at around 9-13 months <ref name="Roos_2018_2" /><ref name="curtain_asbc_2018" />(~26 minutes in). Additional attenuation occurs very slowly for another 7-18 months. De Roos et al. (2018) reported a gradual drop from 4 Plato to 0.5 °Plato during the maturation phase. <ref name="Roos_2018_2" />. During this extended maturation phase, ''Brettanomyces'' continues to ferment the residual sugars leftover in the beer using intra- and extracellular alpha-glucosidase, and produces most of the final aromatic and flavor compounds in the form of esters, phenols, and fatty acids found in finished Belgian lambic and other spontaneously fermented beers (see [[Brettanomyces#Brettanomyces_Metabolism|''Brettanomyces'' metabolism]]). The most abundant species of ''Brettanomyces'' found in spontaneously fermented beer are strains of ''B. bruxellensis'' (''B. lambicus'' is often found, but has been reclassified as a strain of ''B. bruxellensis''). During the maturation phase, a [[pellicle]] is formed from the ''Brettanomyces'', as well as oxidative yeasts from the genera ''Pichia'', ''Candida'', ''Cryptococcus'', and ''Torulspsis'' <ref name="Van Oevelen et al., 1977" /><ref name="Roos_2018" /><ref name="Roos_2018_2" />. It is thought that the pellicle and the presence of these oxidative yeasts might reduce oxygen influx, and thus assist in inhibiting the growth of acetic acid bacteria <ref>[https://pdfs.semanticscholar.org/8c12/9985b9f1264179fe2e2f779bae1ff3e51a54.pdf Jacques De Keersmaecker. "The Mystery of Lambic Beer". Scientific American, Inc. 1996.]</ref>, however, this has not been proven in a scientific manner that we know of.