Changes

Scientific research in Belgium and the US has shown a regular general pattern to the microbial succession of spontaneous fermentation beer. <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" />. This has been illustrated well by Raj Apte <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). The diversity in 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'', ''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 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 Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>. Acidifying the wort to a pH below 4.5 before cooling and exposing to ambient microbes in a coolship can eliminate the enterobacteria phase of spontaneous fermentation, which is a practice for some lambic breweries <ref name="Spitaels et al., 2015" />. 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" />. Wort or beer fermenting during this stage that includes enterobacteria should not be consumed due to 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>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 sp.'' species (predominantly ''S. cerevisiae'' , ''S. bayanus'', and ''S. bayanuspastorianus'', the latter often being present towards the end of this phase). Most of the attenuation is accomplished during this stage, which lasts approximately 3-4 months . 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.

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 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 (note that many scientific publications often use the nomenclature ''Dekkera'' rather than ''Brettanomyces'') <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]). 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). 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 intermediate step) and acetoin from lactic acid and are found on the surface of the wort where oxygen is available. The wort/air interface is also where higher concentrations of acetic acid 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" />.