Changes

'''Spontaneous Fermentation''', for the purposes of this article, refers to the inoculation of wort for fermentation with local ambient microbes. There is a long precedence of this term being used by Belgian lambic producers to describe the part of the lambic brewing process where yeast and bacteria inoculate their wort, and the term has been adopted by commercial brewers in other parts of the world to refer to this process <ref>[https://youtu.be/OBrRPbdCln4?t=4m Pierre Tilquin. Youtube interview. 09/16/2013. Retrieved 10/01/2018. ~4 minutes.]</ref><ref>[https://youtu.be/m_OJv5O8YL8?t=2m26s Jean Van Roy. Youtube interview. 03/13/2014. Retrieved 10/01/2018. ~2:26.]</ref><ref>[https://methodetraditionnelle.org/standards/ Méthode Traditionnelle standards. Retrieved 10/01/2018.]</ref>. Spontaneous fermentation is commonly achieved by use of open [[File:Tilquin blowoff tubes.jpeg|400px|thumb|right|Lambic fermenting at Tilquin with blowoff tubes]]cooling such as in a [[coolship]] where the wort is left exposed to the air and allowed to cool naturally overnight and autochthonous (native) yeast and bacteria are introduced into the wort as it cools. While spontaneous fermentation is part of the traditional brewing process for [[Lambic]] <ref>[http://lambicandwildale.com/the-mystery-of-lambic-beer/ The Mystery of Lambic Beer. Jacques De Keersmaecker. Aug 1996. Retrieved 05/05/2015.]</ref><ref name="Roos_2018_2">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252343/ Wort Substrate Consumption and Metabolite Production During Lambic Beer Fermentation and Maturation Explain the Successive Growth of Specific Bacterial and Yeast Species. Jonas De Roos, Peter Vandamme, and Luc De Vuyst. 2018. DOI: 10.3389/fmicb.2018.02763.]</ref>, not all spontaneously fermented beers necessarily use other processes that lambic production methods use, and Belgian lambic producers insist that the term "lambic" should only be used for beers brewed in Belgium using the various lambic brewing methods (see [[Lambic#Lambic_outside_of_Belgium.3F|Lambic outside of Belgium]]). Spontaneously fermented beers outside of Belgium have been given names such as "spontaneous ales" <ref>[http://www.blackprojectbeer.com/report/2015/1/28/spontaneous-vs-wild "Spontaneous vs. Wild". Black Project website. 01/28/2015. Retrieved 12/26/2018.]</ref><ref>[https://russianriverbrewing.com/beatification "Beatification". Russian River website. Retrieved 12/26/2018.]</ref>, "spontaneous wild ales" <ref>[http://www.degardebrewing.com/events.html De Garde Brewing website. Retrieved 12/26/2018.]</ref>, "Coolship beers" <ref>[https://www.allagash.com/coolship "Brewing With A Coolship: The Science and Art of Spontaneous Fermentation". Allagash Brewing Company website. Retrieved 12/26/2018.]</ref>, with the term "American Coolship Ales" being the adopted term thus far in brewing science for spontaneously fermented beer produced in the Unitied States <ref name="Roos_2018_2" /><ref name="Bokulic et al., 2012">[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035507/ Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale. Bokulich et al, 2012]</ref><ref>[https://www.researchgate.net/publication/341353855_The_power_of_sour_-_A_review_Old_traditions_new_opportunities Bossaert, Sofie, et al. “The Power of Sour - A Review: Old Traditions, New Opportunities.” BrewingScience, vol. 72, no. 3-4, 2019, pp. 78–88.]</ref><ref>[https://www.academia.edu/19646963/Brettanomyces_Bruxellensis_Essential_Contributor_in_Spontaneous_Beer_Fermentations_Providing_Novel_Opportunities_for_the_Brewing_Industry Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry. Jan Steensels. BrewingScience, Sept/Oct 2015 (Vol. 68). 2015.]</ref>. Spontaneous fermentation should not be confused with the various methods of [[Wild_Yeast_Isolation|culturing wild yeast and bacteria]] because many of the microbes that might make a flavor impact during spontaneous fermentation are killed off during the wild yeast culturing processes. Spontaneous fermentation should also not be confused with [https://byo.com/mead/item/1211-open-fermentation-tips-from-the-pros "open fermentation" or "open-top fermentation"], which is a general method of fermenting many styles of beer including English ales and lagers in a vessel that is not closed to the atmosphere.

A [[coolship]] is an open vessel used to cool wort by exposure to ambient air which traditional spontaneous fermentation brewers use to both cool their wort and to inoculate the wort with ambient microbes during the open overnight cooling (8-16 hours; extended cooling times of more than a day might lead to mold growth <ref>[https://www.facebook.com/JesterKingBrewery/posts/10154502699393649?comment_id=10154504389923649&reply_comment_id=10154512163043649&comment_tracking=%7B%22tn%22%3A%22R2%22%7D Thread on Jester King Brewery Facebook thread. 01/16/2017.]</ref>). Traditionally, a coolship is a broad, open-top, flat vessel in which wort cools overnight. The high surface to volume ratio allows for more efficient cooling, which is important at commercial production scales. In addition, this broad, shallow design maximizes the area of wort available for inoculation with ambient microbes. On a homebrew scale, where typical batch sizes cool more quickly, a wide shallow pan is not necessary to achieve appropriate cooling overnight given sufficiently low nighttime outdoor temperatures and the use of a wide shallow pan might result in cooling at a much more rapid rate than seen in traditional commercial production. Boil kettles and similarly shaped vessels are sufficient for overnight cooling for most homebrew batch sizes and may provide a rate of cooling more similar to that provided by coolships in commercial production sized batches <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1068826853145528/ Facebook post by James Howat] </ref>. Cantillon targets a cooled wort temp of 18-20 C (64.4-68 F) after the overnight cooling <ref name="Spontaneous Sour Hour" /> (~50 min in). Traditional producers only carry out spontaneous fermentation between fall and spring when nighttime temperatures have a low of -3.9 to 8°C (25-46°F) to appropriately cool the wort overnight <ref>Conversation between Dave Janssen and Armand Debelder of [[3 Fonteinen]], July 2011</ref><ref name="Spontaneous Sour Hour" />(~39 minutes in, ~54 minutes in). The ambient microbial balance may also be more favorable during this time of year (--some sources say there are more acetic acid bacteria in summer--), but inadequate cooling could result in similar results of enhanced acid production (similar to the effect of warm incubation in [[Wort Souring]], see also [[Spontaneous_Fermentation#Alternative_Applications_of_Spontaneous_Fermentation|Alternative applications of ''Spontaneous Fermentation'']] below). Whatever the root of the different resulting beers based on time of season/ambient nighttime temperature, producers do report different times of year/temperatures exerting a strong influence on the final beer, for example Rob Tod from Allagash Brewing reported solventy and ethyl acetate issues in beers after they were cooled over night at warmer temperatures and aged for 2 years <ref name="Spontaneous Sour Hour" />(~39 minutes in, ~54 minutes in). Russian River Brewing Company reported that cooling the wort before moving it into the coolship results in softer acidity <ref>[https://beerandbrewing.com/podcast-episode-247-spontaneous-brewing-round-table/ Vinnie Cilurzo. Craft Beer and Brewing Magazine Podcast episode #147. 07/12/2022. Retrieved 07/17/2022.]</ref>(~27 mins in).

Some industrial producers of Belgian lambic as well as smaller North American brewers employing spontaneous fermentation acidify their wort to around 4.5 pH before primary fermentation. This was done traditionally by acidifying a portion of wort and adding it to the mash <ref>[https://www.stitcher.com/show/craft-beer-brewing-magazine-podcast/episode/226-for-cantillons-jean-van-roy-brewing-comes-naturally-90577613 Jean Van Roy. Interview with Craft Beer and Beer Magazine Podcast. Episode #226. 02/18/2022.]</ref>(~35 mins in). This may eliminate the enteric bacteria step <ref name="Spitaels et al., 2015" /> (see below, [[Spontaneous_Fermentation#Microbial_Succession_During_Fermentation|Microbial Succession During Fermentation]]). In addition it may act as a safeguard against ''Clostridium botulinum'' (the bacterium responsible for botulism poisoning, which is a serious concern in the food industry because of its high level of toxicity; see [https://www.mayoclinic.org/diseases-conditions/botulism/symptoms-causes/syc-20370262 this Mayo Clinic article]). ''Clostridium botulinum'' can grow at the typical pH range of unfermented and unacidified wort and its spores can survive the boiling process <ref name="James Howat presentation at NHC 2015">[http://www.ahaconference.org/seminars/wild-and-spontaneous-fermentation-at-home James Howat presentation at NHC 2015]</ref>. The degree of botulism risk is not known, though if any reported cases of botulism poisoning from beer exist they are not known to us. Traditional lambic producers have been fermenting unacidified and spontaneously inoculated wort for decades to centuries, which suggests that the risk, if it does exist at all, is very small when following traditional lambic production methods. Furthermore, hops have antimicrobial properties against gram positive bacteria <ref>[http://www.sciencedirect.com/science/article/pii/S0168160503001533/ Sakamoto and Konings, 2003. Beer spoilage bacteria and hop resistance.]</ref> and ''Clostridium botulinum'' is gram positive <ref>[https://en.wikipedia.org/wiki/Clostridium_botulinum/ Clostridium botulinum Wikipedia page]</ref>. The degree to which Hop acids might also partially inhibit ''Clostridium botulinum'' might be resistant to the antimicrobial properties and some other gram-positive pathogens <ref>[https://patents.google.com/patent/US6251461B1/en Antimicrobial activity of hops is unknownextract against Clostridium botulinum, Clostridium difficile and Helicobacter pylori. Eric A. Johnson and Gerhard J. Haas. Google Patents (expired). Filed 1997-10-10. Retrieved 05/27/2022.]</ref>. Some suggest eliminating any potential worry of botulism by acidifying your wort before inoculation <ref name="James Howat presentation at NHC 2015">[http://www.ahaconference.org/seminars/wild-and-spontaneous-fermentation-at-home James Howat presentation at NHC 2015]</ref><ref>[http://suigenerisbrewing.blogspot.com/2017/01/fact-of-fiction-can-pathogens-survive.html "Fact of Fiction - Can Pathogens Survive in Beer? The RDWHAHB Edition". Bryan of Sui Generis Blog. 01/05/2017. Retrieved 01/16/2017.]</ref>. Whether or not this protects from botulism, it will influence the final beer by preventing enteric bacteria growth. In addition, acidifying may influence the activity of ''Pediococcus'' in a spontaneously fermented beer, including the development of "sick" beer, and may therefore alter the final beer (acidic conditions can trigger exopolysaccharide production in some strains of lactic acid bacteria; see [[Pediococcus#.22Ropy.22_or_.22Sick.22_Beer|''Pediodoccus'']]) <ref name="Spontaneous Sour Hour" /> (~1:10 in).

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). '''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.'''  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), as well as a significant population of ''Komagataeibacter'', a genera normally found in kombucha, after 135 day <ref name="Curtin_2021">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2020.1795607?journalCode=ujbc20 Avi Shayevitz, Keisha Harrison & Chris D. Curtin (2021) Barrel-Induced Variation in the Microbiome and Mycobiome of Aged Sour Ale and Imperial Porter Beer, Journal of the American Society of Brewing Chemists, 79:1, 33-40, DOI: 10.1080/03610470.2020.1795607.]</ref>.  Acetic acid bacteria (AAB) are also present during the first stage of fermentation before alcoholic fermentation begins. These consist of a large diversity of species from ''Acetobacter'' and ''Gluconobacter'', with different species thriving more than others at different points during the long fermentation of lambic and some species found being different in different casks <ref name="De_roos_AAB_2018">[https://journals.asm.org/doi/10.1128/AEM.02846-17 Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role. ASM Journals. Applied and Environmental Microbiology. Vol. 84, No. 7. DOI: https://doi.org/10. 1128/AEM.02846-17.]</ref>, including two species that were first described by studies researching lambic (''Acetobacter lambici'' and ''Gluconbacter cerevisiae'' sp. nov. <ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.057315-0 Acetobacter lambici sp. nov., isolated from fermenting lambic beer. Spitaels, Freek and Li, Leilei and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.057315-0.]</ref><ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.059311-0 Gluconobacter cerevisiae sp. nov., isolated from the brewery environment Free. Spitaels, Freek and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.059311-0.]</ref>, as well as ''Acetobacter cerevisiae'' and ''Acetobacter faborum'' <ref name="Roosa_2024">De Roos, J., Vermotea, L., Cnockaertb, M., Vandammeb, P., Weckxa, S., & De Vuysta, L. WOODEN BARRELS HELP TO STEER THE LAMBIC BEER FERMENTATION AND MATURATION PROCESS.</ref>). Acetic acid bacteria are able to grow for the first few weeks because oxygen is available from filling the casks. Once alcoholic fermentation begins, oxygen becomes limited, and the acetic acid bacteria population greatly decreases. Acetic acid bacteria appear again after the alcoholic fermentation phase <ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. For example, Curtin et al. (2018) reported 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). De Ross et al. (2018) reported finding small amounts of acetic acid bacteria in lambic during the first few days of fermentation, which then disappeared once alcoholic fermentation began. AAB then reappeared in the casks in greater numbers at week 7 of fermentation, and continued to be isolated in gradually decreasing cell counts for 24 months, the end of which AAB was no longer isolated <ref name="De_roos_AAB_2018" />.  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 common 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]). Landschoot et al (2015) sampled lambic wort that was pre-acidified to a pH of 4 after being housed in a coolship overnight and during the early weeks of fermentation and found no ''Enterobacteriaceae'Wort or ' in the samples <ref name="Landschoot_2015">[https://www.academia.edu/22769494/The_microbial_diversity_of_an_industrially_produced_lambic_beer_shares_members_of_a_traditionally_produced_one_and_reveals_a_core_microbiota_for_lambic_beer_fermentation?email_work_card=view-paper Spitaels, F., Wieme, A. D., Janssens, M., Aerts, M., Landschoot, A. V., Vuyst, L. D., & Vandamme, P. (2015). The microbial diversity of an industrially produced lambic beer fermenting during this stage should not be consumed due to the fact that some shares members of these are pathogenic bacteria a traditionally produced one and pose potential health risksreveals a core microbiota for lambic beer fermentation. Food Microbiology, 49, 23–32. https://doi.org/10.1016/J.FM.2015.01.008.]</ref>.''' 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 reported that in a spontaneously fermented beer at his commercial brewery 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'', ''S. kudriavzevii'', and ''S. pastorianus'', the latter two species often being present towards the end of this phasein lambic due to the colder cellar temperatures during the winter season when lambic is made). ''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<ref name="Roosa_2024"/><ref name="Bongaerts_2021" />. 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. cerevisiaeSaccharomyces''species, 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 ''KazachsaniaS. kudriavzevii'' 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 is capable 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 breaking down maltooligosaccharides (both homebrewers and professionalsdextrins) have observed yeast fermentation activity typically at 3through alpha-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>. Howeverglucosidase enzyme production, 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 pretherefore can out-acidified to a pH of 4compete ''S.5, and after an initial drop cerevisiae'' 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 later portion of the organic acids that were produced during the initial enterobacteria phase second stage <ref name="Roos_2018_2Bongaerts_2021" />.

The ''[[Saccharomyces]]'' dominated stage of fermentation is followed by prolonged Ethanol, methyl-1-butanol, and gradual succinic acid and flavor development accompanied by are the final points of attenuation, which main compounds produced during this stage for wort that has been pre-acidified. This stage lasts anywhere from 2 to 10 approximately 3-4 months <ref name="Roos_2018" />. In some descriptions this is split into an "acidification phase" which is dominated by lactic acid bacteria (LAB), primarily One study also found populations of ''[[Pediococcus]]Kazachsania'' yeast species and sometimes ''[[Lactobacillus]]'', and a "maturation phase" driven by ''[[Brettanomyces]]Cellulosimicrobium'' yeast species early on in the second stage <ref name="Van Oevelen et al., 1977Roos_2018_2" />. Other sources describe these as one extended maturation phase with acidification from ''Pediococcus'' and ''Brettanomyces'' growth occurring simultaneously <ref name="Spitaels et al., 2015Bongaerts_2021" /><ref name="Bokulic et al. In addition to the bulk of the overall ethanol production, 2012" />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="Spitaels Van Oevelen et al., 20141977" />. When the wort is pre-acidified, the acidification and maturation phases overlap <ref name="Roos_2018" />. Other yeasts such It has been reported by some brewers that this stage might begin as early as ''Candida'', ''Cryptococcus''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 ''Torulopsis'' species have also been isolated from mature lambic, although their impact other faster than possibly being involved reported in the formation of a pellicle is unknown other literature <ref>[httpshttp://onlinelibrarywww.wileyspontanmanc.com/doi/abs/10co.1002uk/j.2050-0416.1977.tb03825.x MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE?p=66 Zach Taylor of Chorlton Brewing Co. D"The Lab Work Begins". Van Oevelen MSpontanmanc blog. Spaepen P08/01/2018. Timmermans HRetrieved 08/29/2018. Verachtert]</ref>. 1977. DOI: MTF members (both homebrewers and professionals) have observed yeast fermentation activity typically at 3-7 days <ref>[https://doiwww.facebook.orgcom/events/10.1002666424196868756/jVarious MTF members.2050 Milk the Funk -0416Collaboration Brew #3: Spontaneous.1977 05/01/2017.tb03825.x Retrieved 08/29/2018.]</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 [httphttps://www.archaicpursuitfacebook.com/2018groups/MilkTheFunk/permalink/081571139996247542/2017-coolship-experiment-hopping-rate.html?mcomment_id=1 this graph 1571597289535146 Raf Soef, James Howat, Levi Funk. Milk The Funk Facebook thread on how long it takes for detailsyeast 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 gradual increase in glucose, maltosepH of 4.5, and maltotriose from week 7 after an initial drop in pH to month 6 due 3.8 by enterobacterial and acetic acid bacteria, the pH rose to 4.0 during the secondary fermentation phase, indicating that the degradation yeast consumed some of maltooligosaccharides (higher chain sugars) the organic acids that were produced during the initial enterobacteria phase <ref name="Roos_2018_2" />.

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''. The species diversity of these genre is lower than during the primary stage due to acid and ethanol selecting for species that are more tolerant to these harsher conditions. For example, De Roos et al. (2018) reported high numbers of ''Acetobacter pasteurianus'', which contains extra genes that code for acid and ethanol tolerance more so than other species of ''Acetobacter'', in lambic from month 3 to month 6, with it disappearing around month 9-13 as ''Pediococcus damnosus'' took its place. ''Acetobacter lambici'' is another species found in lambic during this stage and is well adapted to the lambic environment due to its ability to break down maltooligosaccharides (dextrins) via maltooligosyl trehalose synthase. 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 , ethyl acetate, 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_2" /><ref name="Roos_2018" /><ref name="Bongaerts_2021" />. 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 alDuring a second phase of growth of acetic acid bacteria starting at week 7 in lambic casks, significantly more acetoin (moldy/must flavor when above 50 ppm <ref>[https://www.morebeer.com/articles/Fatty_Flavors_Diacetyl "Fatty Flavors and Diacetyl - Should Your Beer Be Fat-Free?". MoreBeer website. Scott Bickham. Retrieved 04/04/2023. (2018]</ref>) reported high numbers was found in the top portion of ''Acetobacter pasteurianus'' from month 3 the lambic casks above flavor threshold. Acetic acid bacteria has been shown to month 6reduce lactic acid into acetoin, with it disappearing around month 9-13 as ''Pediococcus damnosus'' took its placeand in another study by De Roose et. They also reported finding significant levels of al (2023) the researchers found that ''Acetobacter orientalislambici'' during week 2 and 3 of present in lambic samples that was pre-acidified were tested had the genetic capability to utilize lactic acid as a food source <ref name="Roos_2018_2Roosa_2024" />. Curtin et al. Acetoin was gradually reduced (2018presumably metabolized by ''Brettanomyces'') showed that acetic acid bacteria came and went below flavor thresholds at various random points within a month 9 until it reached near 0-4.5 ppm around month period of fermentation 18 <ref name="curtain_asbc_2018De_roos_AAB_2018" />(~26 minutes in).

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(see ''[[Brettanomyces]]'' for more information), as well as lactic acid bacteria from the genera ''[[Pediococcus]]'' and to a lesser extent ''[[Lactobacillus]]'' and certain other yeast species. 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''). ''B. bruxellensis'' was first isolated from English stock ales in 1904 and then first isolated from lambic in 1921 by Belgian researchers Kufferath and Van Laer. ''B. anomalus'' and ''B. custersianus'' have also been found, but to a lesser extent than ''B. bruxellensis''. ''Pichiamembranifaciens'', ''Debaryomyces hansenii'', and ''Wickerhamomyces anomalus'' are examples of other yeast speciesthat have been found to a lesser extent in lambic during the maturation phase <ref name="Van Oevelen et al., 1977" /><ref name="Roos_2018" /><ref name="Roos_2018_2" /><ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. In some but not all lambic, a shift from ''B. bruxellensis'' to ''B. custersianus'' can occur during the end of maturation, which seems to occur in more porous barrels that have more oxygen ingress and correlates with its preference for a more aerobic environment <ref name="Bongaerts_2021" />.  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" /><ref name="Bongaerts_2021" />. 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.

[https://www.sciencedirect.com/topics/medicine-and-dentistry/biogenic-amine Biogenic amines ] are produced by all living things and are present in many fermented foods and beverages. The biogenic amines that are primarily produced in fermented foods are histamine, tyramine, cadaverine and putrescine. These are produced by microorganisms that can decarboxylate amino acids, and thus their levels in some foods (particularly fish and chicken) are one potential indicator of food freshness or microbiological cleanliness. High dosages can lead to health issues associated with food poisoning or allergic-like reactions such as vomiting, headache, respiratory distress, asthma, hypertension, hypotension, and cardiac palpitation. Thus, biogenic amines have been studied intensely. Although there is no evidence that ''S. cerevisiae'' ale yeast strains produce biogenic amines in beer (even when harvested and re-pitched) <ref>[https://www.sciencedirect.com/science/article/abs/pii/0308814695926613 Influence of Saccharomyces cerevisiae var. uvarum on histamine and tyramine formation during beer fermentation. M. Izquierdo-Pulido, J. Font-Fábregas, C. Vidal-Carou. 1994.]</ref>, biogenic amines in spontaneously fermented beers are produced mostly by enterobacteria. Lactic acid bacteria and yeasts can also produce them to a smaller degree, although a small number of strains of lactic acid bacteria can also degrade biogenic amines. Wort that is pre-acidified greatly reduces the production of biogenic amines, but small levels can still be found. These levels are below the levels found in cheese and fermented sausage and are well below the levels that are acceptable for health <ref name="Wade_2018">[https://onlinelibrary.wiley.com/doi/full/10.1111/ajgw.12366 Role of Pediococcus in winemaking. M.E. Wade, M.T. Strickland, J.P. Osborn, C.G. Edwards. 2018. DOI: https://doi.org/10.1111/ajgw.12366.]</ref><ref name="loret_2005">[https://www.sciencedirect.com/science/article/abs/pii/S0308814604002365 Levels of biogenic amines as a measure of the quality of the beer fermentation process: Data from Belgian samples. S. Loret, P. Deloyer, G. Dandrifosse. 2005.]</ref><ref>[https://www.academia.edu/13219732/Biogenic_Amines_Degradation_by_Lactobacillus_plantarum_Toward_a_Potential_Application_in_Wine?email_work_card=title Capozzi, Vittorio et al. “Biogenic Amines Degradation by Lactobacillus Plantarum: Toward a Potential Application in Wine.” Frontiers in Microbiology 3 (2012): n. pag. Web.]</ref>.

Loret et al (2005) examined the levels of biogenic amines in Belgian beers, including lagers, traditional ales, bottle conditioned ales, and spontaneously fermented beer (presumably lambic). They found that spontaneously fermented beers, which were 42 samples out of the total 297 samples and from 10 different breweries, generally contained the most biogenic amines, namely tyramine (associated with hypertension), histamine (associated with hypotension), and cadaverine. However, not all of these breweries produced higher levels of amines, suggesting that processing in some breweries is limiting the biogenic amine production (this process was not identified in the Loret et al study, but the process is likely to be the lowering of the wort pH to 4.5 to limit the enterobacteria phase). For example, 6 of the spontaneous fermentation breweries had levels of histamine as an average between 20-45 mg/l, and levels of tyamine between 30-60 mg/l, which is higher than the upper limit that is generally considered to be safe for consumers (this upper limit was stated as 10 mg/l by Loret et al.). The other 4 breweries had levels of histamine and tyramine between 0-20 mg/L, as an average. Interestingly, they also found that a small number of ales and bottle conditioned ales, 21 out of 220 samples, also had levels of tyamine above the upper limit of 10 mg/l. None of the lagers sampled had biogenic amines above 10 mg/l <ref name="loret_2005" />. Another study in Portugal measured biogenic amines in 5 Portuguese craft beers (4 ales and 1 lager; no mixed fermentation or sour beers) and reported that the total biogenic amine content was less than 10 mg/l for all of the beers <ref>[https://www.mdpi.com/1424-8220/23/1/343 Gil, R.L.; Amorim, C.M.P.G.; Amorim, H.G.; Montenegro, M.d.C.B.S.M.; Araújo, A.N. Influence of Brewing Process on the Profile of Biogenic Amines in Craft Beers. Sensors 2023, 23, 343. https://doi.org/10.3390/s23010343.]</ref>.  De Roos et al. (2018) measured biogenic amines over the fermentation lifespan of Belgian lambic beers that were pre-acidified to a pH of 4.5 before being cooled in a coolship and spontaneously fermented. They found that the initial wort had low concentrations of some biogenic amines, such as agmatine (9 mg/l), putrescine (8 mg/l), and cadaverine (3 mg/l). In one cask, the agmatine remained stable while in the second cask the agmatine declined to zero during the maturation phase, and then slightly increased to less than 5 mg/l. Cadaverine was produced during the first three weeks of fermentation and remained steady throughout the fermentation process at about 30 mg/l. Histamine was produced during the acidification phase by ''Pediococcus damnosus'' between 3 and 9 months and ended up at around 15 mg/l. Tyramine had final concentrations of around 30-40 mg/l and was formed either during the acidification phase (6 months) or the late maturation phase (18-24 months), potentially by ''P. damnosus'' or some other LAB that was at too low of a population to detect, or maybe as a result of autolysis of dead yeast cells. 2-Phenylethylamine and tryptamine were never found in the lambic beers <ref name="Roos_2018_2" />. A second study by the same group of researchers in 2024 confirmed that ''P. damnosus'' was responsible for producing histamine in lambic samples that were tested at one brewery; the total biogenic amines remained below the regulatory levels deemed safe for consumption in Europe <ref name="Roosa_2024"/>.

De Roos et al. (2018) measured The biogenic amines over the fermentation lifespan content of Belgian lambic beers spontaneously fermented beer that were pre-is not acidified before fermentation is not likely to a pH be high enough to cause serious physical harm. Large amounts of 4.5 beer must be consumed before being cooled in a coolship and spontaneously fermentedadvisable limits are reached. They found that However, the initial wort had low concentrations additive effect of different amines consumed with a large amount of beer might cause health related reactions in some biogenic amines, such as agmatine (9 mgpeople. See [http:/l), putrescine (8 mg/l), and cadaverine (3 mgsuigenerisbrewing.com/l)index. In one cask, the agmatine remained stable while in the second cask the agmatine declined to zero during the maturation phase, and then slightly increased to less than 5 mgphp/2019/l. Cadaverine was produced during the first three weeks of fermentation and remained steady throughout the fermentation process at about 30 mg01/l. Histamine was produced during the acidification phase by ''Pediococcus damnosus'' between 3 and 9 months and ended up at around 15 mg22/l. Tyramine had final concentrations of around 30biogenic-40 mgamines/l and was formed either during the acidification phase (6 months) "Fact or the late maturation phase (18-24 months), potentially Fiction – Biogenic Amines in Beer" by ''PDr. damnosus'' or some other LAB that was at too low Bryan Heit; an analysis of a population to detect, or maybe as a result of autolysis of dead yeast cells. 2-Phenylethylamine biogenic amines in spontaneously fermented beer and tryptamine were never found in the lambic beers <ref name="Roos_2018_2" />associated health concerns] for more information.

The sources of microbes that influence spontaneous fermentation are somewhat debatable, especially when referencing [[Lambiclambic]] producers in Belgium. Some Belgian lambic brewers have made claims historically that microbe populations in the valley of the Senne River are unique enough that spontaneously fermented beer cannot be made anywhere else; however, American Coolship Ales and scientific inquiry have demonstrated that the genus level microbes that ferment spontaneously fermented beer are very similar in other parts of the world (although species and strain differences might have an impact on specific flavor profile differences between different regions of the world) <ref name="curtain_asbc_2018" /><ref>[https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035507 Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale. Bokulich NA, Bamforth CW, Mills DA (2012) Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale. PLOS ONE 7(4): e35507. https://doi.org/10.1371/journal.pone.0035507]</ref>. Although the coolship step is regarded by lambic brewers as the main contributor to the microbial inoculation for spontaneous beers, it has been demonstrated that ''Brettanomyces'' and other microbes living in the barrels is are at least partially responsible for the secondary fermentation of spontaneous beers. Some yeast species that have been found in limited capacity in lambic such as ''Wickerhamomyces anomalus'' and ''Debaryomyces hansenii'' have been isolated from the air of coolship rooms, but they have also been isolated from the air of cellar rooms, indicating that they could have been introduced to the wort/beer in the cellar as well. Additionally, yeast and bacteria species that are the most abundant during lambic fermentation (''Saccharomyces'', ''Brettanomyces'', ''Pediococcus'', and acetic acid bacteria) have not been isolated from the air of coolship rooms <ref name="Bongaerts_2021" />. De Roos et al. (2018) used amplicon sequencing technology (which is better at detected so-called "[[Quality_Assurance#Viable_But_Nonculturable|viable but not culturable]]" cells) to sample of the interior of barrels and foeders used in one lambic brewery that uses high-pressurized hot water and sulfur dioxide to clean them, and found surviving colonies of ''Brettanomyces anomalus'', ''B. bruxellensis'', ''B. custersianus'', ''Pediococcus'', and to a lesser extent ''Acetobacter'' and ''Lactobacillus''. The ''Acetobacter'' might be linked to older barrels that are more porous. ''Pichia'', ''Debaryomyces hansenii'' and ''Candida'' were also found in abundance in some barrels, but not others. They also found high levels of ''Cellulosimicrobium'' and ''Acinetobacter'', which have not been found to be important for lambic fermentation; the authors suspected that these were living in the wood rather than in the beer since they can metabolize cellulose. Although they are probably also not important to the fermentation of lambic, molds were found in the more porous barrels before cleaning, including ''Aspergillus'' and ''Penicillium''. No molds survived the sulfuring process and the diversity of microbes was far higher in barrels than it was for foeders, perhaps because of the higher level of oxygen exposure in barrels compared to foeders, although foeders had a much higher than expected amount of surviving ''Saccharomyces'' and ''Pichia'' <ref>[https://aem.asm.org/content/early/2018/10/15/AEM.02226-18 The interior surfaces of wooden barrels are an additional microbial inoculation source for lambic beer production. J. De Roos, D. Van der Veken, L. De Vuyst. 2018. DOI: 10.1128/AEM.02226-18.]</ref>.  Professor Chris Curtin from Oregon State University presented the results of a study by his team where 50 barrels of three different vintages of spontaneously fermented, lambic-inspired beer was sampled three times over a 9 month period. These 50 barrels represented three batches of the same recipe brewed in 2013, 2015, and 2016. With the exception of a small number of barrels, they all contained the same or similar ''Brettanomyces bruxellensis'' yeast. There was no significance between the three different batches. Two of the 2015 barrels had a different species of ''Brettanomyces'' (''claussenii''). Two of the 2016 barrels had more ''Saccharomyces cerevisiae''. This study indicated that barrel to barrel variation as far as yeast goes is fairly small at this brewery. Bacteria populations clustered much closer based on the vintage (but not the barrel). The 2013 batch was dominated by ''Gluconobacter'' and ''Acetobacter'', while the 2015 batch was dominated only by ''Acetobacter'', and the 2016 batch was dominated by ''Lactobacillus''. This indicated that the vintage of the batch plays a major role in determining which bacteria will be the dominant bacteria during maturation, but individual barrels generally do not (although a small number of barrels matured faster than the majority of the barrels and were dominated by bacteria that represented the more mature vintage). Despite the lack of major variation between barrels, Curtin determined that some barrels can introduce microbial variation, perhaps due to insects transferring microbes or differences in the oxygen ingress between different barrels, and the ability of yeast and bacteria to live within the inner surface of the barrels and potentially survive cleaning procedures <ref name="curtain_asbc_2018" /> (~29 minutes in)<ref name="Curtin_2021" />.

De Roos Landschoot et al. (2018) used amplicon sequencing technology (which is better at detected so-called "[[Quality_Assurance#Viable_But_Nonculturable|viable but not culturable]]" cells2015) attempted to samples of recover yeasts and bacteria sampled from the interior of barrels brewery walls, ceilings, and foeders used coolship in one lambic an industrial brewery in West-Flanders that uses high-pressurized hot water and sulfur dioxide also produces lambic. They were unable to clean them, and found surviving colonies recover any yeasts or bacteria from these surfaces. The team did recover several species of microbes from the air of the coolship room such as ''Brettanomyces anomalusKlebsiella oxytoca'', ''B. bruxellensisBacillus'', and ''B. custersianusStaphylococcus'', ''Pediococcus''but these microbes were not found in the wort after cooling in the coolship nor in the foeders during the sample times of 1, 2 and to a lesser extent 3 weeks. ''AcetobacterS. cerevisiae'' and ''Lactobacillus, ''S. The pastorianus''Acetobacter, '' might be linked to older barrels that are more porousD. ''Pichiabruxellensis'', ''Debaryomyces hanseniiP. damnosus'' , and ''Candida'' a diverse range of acetic acid bacteria were also the dominate species of microbes found during the fermentation of lambic in abundance in some barrelsthis brewery. After boiling, but not othersthe brewery cooled its wort to 40°C using an industrial heat exchanger before pumping it into the coolship to reside overnight for 24 hours. They also found high levels of ''Cellulosimicrobium'' The foeders in this brewery are cleaned with a pressure washer and ''Acinetobacter'', which have not been found are no attempt was made to be important for lambic fermentation; pasteurize or sanitize the foeders with heat or chemicals. The inside surfaces of the authors suspected that foeders were sampled and these samples were living in the wood rather than in the beer since they can metabolize cellulose. Although they are probably also not important to only samples that contained microbe species that were found during the fermentation of the lambic, molds were found in . The authors concluded that the more porous barrels before cleaning, including ''Aspergillus'' and ''Penicillium''. No molds survived microbes fermenting lambic at this brewery are introduced into the sulfuring process and wort via the diversity of microbes was far higher in barrels than it was for foeders, perhaps because wooden inner surfaces of the higher level of oxygen exposure in barrels compared to foeders, although foeders had a much higher than expected amount of surviving ''Saccharomyces'' and ''Pichia'' <ref>[https://aem.asm.orgname="Landschoot_2015"/content/early/2018/10/15/AEM.02226-18 The interior surfaces of wooden barrels are an additional microbial inoculation source for lambic beer production. J. De Roos, D. Van der Veken, L. De Vuyst. 2018. DOI: 10.1128/AEM.02226-18.]</ref>.

Professor Chris Curtin from Oregon State University presented the results of De Roos et al. (2024) reported finding a study by his team where 50 barrels very diverse set of three different vintages of spontaneously fermented, lambicspecies in wort sampled from coolships after cooling (100-inspired beer was sampled three times over a 9 month period200 mL samples). These 50 barrels represented three batches Using DNA sequencing techniques and in order of the same recipe brewed in 2013abundance, 2015they found ''H. uvarum'', and 2016''S. With the exception of a small number of barrelscerevisiae'', ''Penicillium roqueforti'', ''Acinetobacter guillouiae'', they all contained the same or similar ''Brettanomyces bruxellensisLactococcus raffinolactis'' yeast. There was no significance between the three different batches. Two of the 2015 barrels had a different species of , ''BrettanomycesGeotrichum candidum'' (, ''clausseniiChryseobacterium bovis''). Two of the 2016 barrels had more , ''Saccharomyces cerevisiaeFlavobacterium hibernum''. This study indicated that barrel to barrel variation as far as yeast goes is fairly small. Bacteria populations clustered much closer based on the vintage (but not the barrel). The 2013 batch was dominated by , ''GluconobacterYarrowia lipolytica'' , and ''AcetobacterTrichococcus flocculiformis''. However, while the 2015 batch was dominated only by species from the coolship samples that were found during fermentation in two Italian wine barrels were ''AcetobacterH. uvarum'', and the 2016 batch was dominated by ''LactobacillusS. cerevisiae''. This indicated The authors concluded that the vintage wooden casks themselves, despite heavy cleaning methods, contributed the majority of the batch plays a major role in determining which bacteria will be the dominant bacteria impactful microbe species during maturationfermentation and aging, but individual barrels generally do not including ''Brettanomyces'' <ref name="Roosa_2024"/>. The authors of the study write (although a small number see also [https://www.facebook.com/groups/MilkTheFunk/posts/7935877029773775/ comments by Dr. Bryan Heit this MTF post]): <blockquote>''The present study further contributed to the role of wooden barrels matured faster than in the majority spontaneous inoculation of the barrels fermenting wort and were dominated by bacteria maturing beer during lambic beer production. Although shotgun metagenomics revealed that represented the more mature vintage). Despite cooled wort had the lack of major variation between barrelshighest diversity and evenness, Curtin determined that some barrels can introduce microbial variation, perhaps due its harboring species did not contribute to insects transferring microbes or differences in the oxygen ingress between different barrels, wort fermentation and beer maturation in the ability of yeast and bacteria to live within the inner surface of the barrels and potentially survive cleaning procedures casks.'' <ref name="curtain_asbc_2018Roosa_2024" /> (~29 minutes in). </blockquote>

Various brewer anecdotes from experiments appear to contradict the published scientific literature. James Howat of Black Project Spontaneous Ales reported conducting an experiment that showed a similar fermentation profile between a barrel fermented spontaneous beer and samples taken from the coolship and aged in glass flasks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1977772602250944/?comment_id=1977929225568615&reply_comment_id=1978520655509472&comment_tracking=%7B%22tn%22%3A%22R%22%7D James Howat. Milk The Funk thread on the source of ''Brettanomyces'' in lambic. 02/05/2018.]</ref>. The spontaneous beers at Black Project were produced for a few years by placing a kettle on the roof outside of the brewery to collect microbes from the air and then racked directly to barrels, so there was no influence of microbes living within the brewery (Black Project now uses a coolship inside the brewery). This led James to conduct this experiment to see how much the barrels were influencing the microbiome of the beers even after they were steamed to the [[Barrel#Sanitizing|point of possible pasteurization]]. James reported that the sensory characteristics between the barrel aged beers and the flask-aged beers were very similar, other than the obvious differences that the oak would have provided, which led him to believe that air inoculation provides a significant contribution to the microbial load of spontaneously fermented beers. Additionally, Brasserie-Brouwerij Cantillon (ref needed) and Oud Beersel <ref>[https://soundcloud.com/craftbeerbrew/podcast-episode-21-new-belgiums-wood-cellar-director-blender-lauren-limbach Lauren Limbach. Craft Beer and Brewing Magazine Podcast. Episode 21. 02/16/2018.]</ref> (~42 minutes in) are known to steam clean their barrels which might be enough to [[Barrel#Sanitizing|sanitize them]], although former Cantillon brewer and saison expert Yvan De Beats maintains that Cantillon barrels are not heated enough to be pasteurized <ref>[https://www.crowdcast.io/e/saison-ale-myths-yvan-baets/1 De Beats, Ybsn. Doug pipers Crowdcast. 08/26/2021.]</ref>. Pierre Tilquin reported that different worts brewed and cooled by different lambic brewers present different fermentation and flavor profiles when barrel fermented in his blendery, particularly when he began steam cleaning emptied barrels <ref>[https://beerandbrewing.com/podcast-episode-234-pierre-tilquin-of-gueuzerie-tilquin-makes-lambic-and/ Pierre Tilquin. Interview on Craft Beer & Brewing Podcast. Episode 234. 04/15/2022.]</ref>(~15 and ~27 mins in). Mitch Ermatinger of Speciation Artisan Ales reported moving wort cooled over night overnight in a coolship to a stainless fermenter, and the wort began showing signs of visual fermentation four days later <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3012223975472463/?comment_id=3012239425470918&reply_comment_id=3012428892118638 Mitch Ermatinger. Milk The Funk Facebook thread on sources for microbes in spontaneous fermentation. 10/28/2019.]</ref>. Such anecdotes deserve further investigation using the full scientific process.

[[Blending]] is a fundamental part of traditional spontaneous beer production (and typically of wood aged sour beer production in general). In barrel aged mixed fermentation beer, and especially spontaneously fermented beer, there is a high potential for variability in different barrels/fermentation vessels, even those resulting from the same hot side process. To help create a more balanced and complex product, producers of sour beers often blend barrels (of both the same and of different vintages) together into one final product. In Belgian gueuze production, two and three year old beer is blended with one year old beer. The three/two year old beer contains fermentative microbes while the younger one year old beer contains residual dextrins. Sucrose is often added to the bottles to ensure adequate carbonation (see [[Packaging]]). Packaged Belgian gueuze can age for decades with aromas and flavors continuing to develop during storage <ref name="Bongaerts_2021" />. The homebrewer can employ the same techniques and blend to reach the desired final product from beers of different vintages and different carboys/vessels of the same brew. See the [[blending]] page for more information on this topic.

Frequently , a non-trivial amount of beer is dumped at spontaneous beer breweries <ref>[https://www.youtube.com/watch?v=QUa0QH6niiQ Sour Beer Panel at the Firestone Walker International Beer Fest]</ref> (~8.5 min in). The exact amount depends on the conditions of the brewery and the willingness of the brewer to try to blend in batches that might not taste as good and/or have mild off flavors at the expense of the overall quality of the blend, but commercial brewers have reported dumping levels of 5% (and possibly up to 15%) of total production <ref name="Beer Temple interview with De Garde">[https://vimeo.com/127084279 The Beer Temple Interviews #264 with Trevor Rogers of De Garde]</ref> (~13 minutes in). This may be due to an imbalance in the microbes <ref name="Beer Temple interview with De Garde"></ref> (~14 minutes in) or a bad barrel resulting in off woody flavor <ref name="Spontaneous Sour Hour" /> (~1:31 in) or excessive O2 exposure. In addition to the beer inside such barrels being dumped, the barrel itself is also often discarded <ref name="Beer Temple interview with De Garde"></ref> (~14 minutes in). Homebrewers who are fermenting spontaneously may expect that from time to time they will need to dump a batch.