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 scientific literature 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 dextrinous wort consisting of a high concentration of maltooligosaccharides may be produced by different mashing procedures. The most traditional method of achieving this is through a [[Turbid Mash|turbid mash]]. With this mashing technique, unconverted [[File:Homebrew Turbid Mash Pull.jpeg|200px|thumb|right|First pull of turbid runnings in a homebrew turbid mash]] starchy wort (which is turbid in appearance) is pulled from the mash and heated to denature enzymes. These pulled runnings are then replaced by infusions of hot water as the mash is carried through a series of steps for conversion of the remaining grains. The starchy wort from the early 'turbid' pulls is carried to the boil with incomplete conversion, providing dextrins to sustain ''[[Brettanomyces]] '' and lactic acid bacteria in a prolonged mixed fermentation. Other methods to carry unconverted dextrins into the boil may be employed such as the addition of flour <ref name="Burgundian Babble Belt discussion">[http://www.babblebelt.com/newboard/thread.html?tid=1108752780&th=1243453104 Burgundian Babble Belt discussion]</ref> passing hot mash runnings through flaked grains <ref name="Flat Tail on the Brewing Network">[http://thebrewingnetwork.com/shows/1027/ Flat Tail on the Brewing Network, ~1:04 in]</ref>, or pulling mash runnings before full conversion without the prolonged processing of a turbid mash <ref name="Flat Tail on the Brewing Network" />. Whichever technique is employed, the goals are the same - to provide starches which ''Saccharomyces cerevisiae'' and ''Saccharomyces pastorianus'' cannot ferment and which can feed the diverse combination of other yeasts and bacteria present.

Although creating a dextrinous wort is traditional for spontaneous fermentation, it is not necessarily a microbiological requirement. Traditional [[Lambic]] must use a turbid mashing process for the sake of tradition, but non-lambic spontaneous fermentations can also be successful using simpler methods such as single infusion mashes. Not having the dextrins available for the microbes during long-term aging might change the fermentation profile and produce a different type of product (for example, with fewer dextrins there might be less acidity produced from the slow acting ''Pediococcus'', but ''Brettanomyces'' does not rely solely on dextrins to produce its flavor contributions and fewer dextrins will accomplish a faster stable final gravity). Many brewers outside of Belgium have experimented with doing spontaneous fermentation with wort that was not produced from a turbid mash (references needed). Additionally, the maltooligosaccharides produced by turbid mashing can be broken down earlier in the fermentation process by other species such as ''Saccharomyces kudriavzevii'' and acetic acid bacteria <ref name="Bongaerts_2021">[https://pubmed.ncbi.nlm.nih.gov/34232060/ Bongaerts D, De Roos J, De Vuyst L. Technological and Environmental Features Determine the Uniqueness of the Lambic Beer Microbiota and Production Process. Appl Environ Microbiol. 2021 Aug 26;87(18):e0061221. doi: 10.1128/AEM.00612-21. Epub 2021 Aug 26. PMID: 34232060; PMCID: PMC8388830.]</ref>.

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 presence of more than at least 2-5 ppm of dissolved oxygen (DO) in the wort might also reduce the risk of botulism <ref name="Pérez-Fuentetaja">[https://link.springer.com/article/10.1007/s10750-005-0011-1 Influence of Limnological Conditions on Clostridium Botulinum Type E Presence in Eastern Lake Erie Sediments (Great Lakes, USA). Alicia Pérez-Fuentetaja, Mark D. Clapsadl, Donald Einhouse, Paul R. Bowser, Rodman G. Getchell, W. Theodore Lee. 2006.]</ref>(more references needed); however, the levels of DO in wort that has been cooled in a coolship has not been well studied, and neither has the amount of DO during the first few days of fermentation. Dissolved oxygen in wort that is near boiling temperatures will be limited due to Hentry's law, but some amount of atmospheric oxygen will be absorbed as the wort cools over night <ref>[https://www.boundless.com/physiology/textbooks/boundless-anatomy-and-physiology-textbook/respiratory-system-22/gas-laws-210/henry-s-law-1032-977/ "Henry's Law". Bouldess.com website. Retrieved 03/07/2017.]</ref><ref>[http://docs.engineeringtoolbox.com/documents/639/oxygen-solubility-water-2.png Graph of oxygen solubility in water at different temperatures. Engineering Toolbox website. Retrieved 03/07/2017.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1599584193403122/?comment_id=1599693336725541&reply_comment_id=1600361503325391&comment_tracking=%7B%22tn%22%3A%22R%22%7D Bryan of Sui Generis blog. MTF discussion on dissolved oxygen in wort cooled in a coolship, and the accuracy of DO meters. 03/02/2017.]</ref>. Some reports of DO in wort cooled in a coolship MTF include ~4 ppm in a small coolship that was 2' x 1' x 1', and 3.6 - 3.8 ppm in wort cooled overnight in an open 10 gallon boil kettle <ref>[https://www.facebook.com/browse/likes?id=1605741916120683 Amaral, Justin. MTF discussion on dissolved oxygen in coolship wort. 03/07/2017.]</ref>. The DO levels from a commercial sized coolship (10 BBL; 6' x 10') were reportedly 2.6 ppm after the transfer to the coolship while the wort was still hot, and 5.1 ppm after the wort cooled for 14 hours <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1601687793192762/?comment_id=1601698063191735&reply_comment_id=1602191886475686&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Coker, Ryan. MTF discussion on dissolved oxygen in wort cooled in a commercial coolship. 03/07/2017.]</ref>. The dissolved oxygen levels could increase during the filling of the barrels, although there is no data on this that we know of currently. The dissolved oxygen in the wort, however, could be quickly consumed by aerobic bacteria and yeast (which then generally produce a low pH environment that is hostile to ''Clostridium botulinum''). Additionally, some strains of ''C. botulinum'' are more oxygen tolerant than others. Therefore, DO levels should not be relied upon for preventing botulism. Instead, either a timely fermentation is desirable (within 4 days has been a suggestion; however , it is not known how long it would take ''C. botulism'' to grow in anaerobic wort and produce enough botulism toxin <ref>[http://beerandwinejournal.com/botulism/ "Storing Wort Runs the Risk of Botulism". Dr. Colby, Chris. Beer and Wine Journal blog. 04/17/2014. Retrieved 03/07/2017.]</ref>. , or reducing the Other conditions that prevent ''C. botulinum'' from growing include a reduction in pH below 4.6 and an increase in alcohol (decrease in gravity) 5 before the wort is cooled or immediately after <ref name="Pérez-Fuentetaja" /><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1604630206231854/?comment_id=1605313322830209&reply_comment_id=1605352169492991&comment_tracking=%7B%22tn%22%3A%22R%2312%22%7D Bryan Heit of Sui Generis Blog. MTF discussion on dissolved oxygen in wort cooled in a coolship. 03/07/2017.]</ref>.

* [http://suigenerisbrewing.com/index.php/2020/01/29/botulism-in-beer/ "Fact or Fiction – Botulism in Beer?" by Dr. Bryan Heit of Sui Generis Brewing] and [http://beerandwinejournal.com/botulism/ "Storing Wort Runs the Risk of Botulism", by Dr. Colby, Chris.]

The In Belgian lambic and non-Belgian lambic-style production, the wooden fermentation vessels are frequently used/flavor neutral oak wine barrels in the 220-400 L (58-105 gal) range but other woods such as chestnut are used and the vessels may also be large tuns or foudres holding upwards of 45 HL (about 1200 gal, or about 34 bbl). These barrels provide two primary benefits for the fermentation - they allow a small amount of oxygen permeability and they can provide an environment which houses some of the microbes active in the fermentation (although there is some debate around this, see [[Spontaneous_Fermentation#Notes_on_the_Source_of_Microbes|Sources for Microbes]] below). Notably, ''Brettanomyces'' could survive some cleaning regimes by penetrating into the wood and in some cases possibly metabolize compounds present in the wood such as cellobiose, which is produced from toasting of the wood <ref name="Vinnie on the Session Jan 2010">[http://www.thebrewingnetwork.com/post1940/ Vinnie Cilurzo of Russian River on the Brewing Network's Sunday Session, 17-January-2010]</ref>(~3:22 in). While a controlled micro-oxidation can be beneficial to the beer, too much oxygen exposure can lead to excessive acetic acid and/or ethyl acetate production (either from ''Brettanomyces'' or ''Acetobacter'') <ref name="yakobson1">[http://www.brettanomycesproject.com/dissertation/pure-culture-fermentation/pure-culture-fermentation-discussion/ Yakobson, Chad. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Pure Culture Fermentation Discussion. 2011.]</ref>. In addition Wooden barrels are usually kept horizontal and full in order to minimize headspace and reduce the growth of aerobic microbes and also reduce the production of acetic acid <ref name="Bongaerts_2021" />. Although wooden barrels used in spontaneous fermetnation production are usually flavor neutral, barrels may still provide flavor and structure from tannins and, in some cases, what they previously held.

On a homebrew scale a fair amount of attention has been paid to the topic of oxygen permeability in different fermentation vessels and closures <ref>[httphttps://www2www.parchomebrewtalk.com/emdl/membersmedia/raj-apte/GingerBeer-o2-table.pdf 58958 Raj Apte's oxygen permeability table]</ref> <ref>[http://www.mocon.com/assets/documents/PPS_Article_highq.pdf Better Bottle closure study]</ref> <ref>[https://www.youtube.com/watch?v=boLqmFIzUZ0&list=PLibE2BjPG_8H0IZe4fS2FD4uidCFhgzBn&index=4 Dan's video discussing airlocks and fermenters]</ref>. It has been suggested that sealing a glass carboy with a wooden dowel or chair leg can result in similar oxygen permeability as a wine barrel. Although this was quite a clever idea for replicating oxygen exposure, this is not recommended as it can lead to breakage of the glass carboys <ref name="Mad Fermentationist Oak">[http://www.themadfermentationist.com/2007/02/8-homebrew-barrel.html Mad Fermentationist $8 homebrew barrel]</ref>. While micro-oxygenation may be an important part of some spontaneous production it may be getting too much attention in homebrew carboy conditions <ref name="Mad Fermentationist Oak">[http://www.themadfermentationist.com/2007/02/8-homebrew-barrel.html Mad Fermentationist $8 homebrew barrel]</ref> (see comments) relative to other controls such as temperature, microbes, and time. See the [[Barrel]] page for discussions on the barrels available to homebrewers. Since spontaneous fermentations can take several days to begin (generally 4-7 days, although we have seen reports of up to two weeks), some professional brewers and a microbiologist have recommended that carboys should be filled as close to the neck as possible to limit the initial headspace and oxygen in that headspace so as to avoid [[mold]] growth (lowering the wort pH to under 4.5 will also help prevent mold growth during the early stages of fermentation) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1247501295278082/?comment_id=1247509875277224&comment_tracking=%7B%22tn%22%3A%22R%22%7D MTF post regarding mold growth in homebrew spontaneous fermentations. 03/06/2016.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1442080252486851/?match=c3BvbnRhbmVvdXMsZmVybWVudGVkLDEwMA%3D%3D MTF post regarding limiting headspace to prevent mold growth. 10/19/2016.]</ref>.

Regarding fermentation temperature, commercial producers looking for balanced acidity and flavor/aroma complexity prefer cooler fermentation temperatures in the range of the high 50s to low 60s F °F (~13-18 Cbetween 10°C and 20°C) <ref name="Spontaneous Sour Hour" /> (~1:14 in)<ref name="Bongaerts_2021" />. Temperature control is very important to some Lambic producers. 3 Fonteinen had temperature controlled cellars, highlighting the importance of aging temperature. Unfortunately the temperature control thermostat failed and resulted in the brewery nearly going out of business <ref>[https://www.lambic.info/Brouwerij_3_Fonteinen lambic.info 3F]</ref>. This temperature range allows slow and balanced fermentation by the diverse array of microbes present. Warming the fermentation too much results in enhanced production of acidity which is out of line with what the lambic producer is aiming for. This can be used to the advantage of the brewer when producing certain non-lambic inspired spontaneously fermented beers (see below, [[Spontaneous_Fermentation#Alternative_Applications_of_Spontaneous_Fermentation|Alternative applications of spontaneous fermentation]]).

===Blending Microbial Succession During Fermentation===[[File:Sofie Bossaert 2019 Fig2.JPG|thumbnail|700px|[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.]]] ====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). '''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 Dumpingwent 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'' 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 shares members of a traditionally produced one and reveals 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 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>. ====Second Stage: Ethanol Production====

[[Blending]] is a fundamental part The second stage of traditional spontaneous beer production fermentation is dominated by ''Saccharomyces'' species (predominantly ''S. cerevisiae'', ''S. bayanus'', ''S. kudriavzevii'', and typically ''S. pastorianus'', the latter two species often being present towards the end of wood aged sour beer production this phase in generallambic due to the colder cellar temperatures during the winter season when lambic is made). In barrel aged mixed ''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 beer, and especially spontaneously fermented beer, there which is a high potential for variability in different barrelscharacterized by ethanol production <ref name="Roosa_2024"/><ref name="Bongaerts_2021" /fermentation vessels>. Most of the attenuation is accomplished during this stage with the depletion of monosaccharides, disaccharides, 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 trisaccharides consumed in that order (of both glucose/fructose is consumed first by the same ''Saccharomyces'' species, and then maltose/maltotriose are gradually depleted until they are gone by the end of different vintagesthe second stage) together into one final product. The homebrewer ''S. kudriavzevii'' is capable of breaking down maltooligosaccharides (dextrins) through alpha-glucosidase enzyme production, and therefore can employ out-compete ''S. cerevisiae'' in the same techniques and blend to reach later portion of the desired final product from beers of different vintages and different carboyssecond stage <ref name="Bongaerts_2021" /vessels of the same brew. See the [[blending]] page for more information on this topic>.

Frequently a nonEthanol, 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-trivial amount 4 months. One study also found populations of beer is dumped at spontaneous beer breweries ''Kazachsania'' yeast species and ''Cellulosimicrobium'' yeast species early on in the second stage <refname="Roos_2018_2" />[https://www.youtube.com/watch?v<ref name=QUa0QH6niiQ Sour Beer Panel at the Firestone Walker International Beer Fest]<"Bongaerts_2021" /ref> (~8.5 min in). The exact amount depends on In addition to the conditions bulk of the brewery and overall ethanol production, this phase also sees the willingness production of the brewer to try to blend in batches that might not taste as good higher alcohols and/or have mild off flavors at the expense synthesis of the overall quality of the blendesters, especially isoamyl acetate, as well as glycerol, caprylic acid, but commercial brewers have reported dumping levels of 5% (and possibly up to 15%) of total production capric acid <ref name="Van Oevelen et al., 1977" /><ref name="Beer Temple interview Roos_2018" />. It has been reported by some brewers that this stage might begin as early as 3-14 days and corresponds with De Garde"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>[httpshttp://vimeowww.comspontanmanc.co.uk/127084279 ?p=66 Zach Taylor of Chorlton Brewing Co. "The Beer Temple Interviews #264 with Trevor Rogers of De GardeLab Work Begins". Spontanmanc blog. 08/01/2018. Retrieved 08/29/2018.]</ref> . MTF members (~13 minutes inboth homebrewers and professionals)have observed yeast fermentation activity typically at 3-7 days <ref>[https://www.facebook. This may be due to an imbalance in com/events/666424196868756/ Various MTF members. Milk the microbes Funk - Collaboration Brew #3: Spontaneous. 05/01/2017. Retrieved 08/29/2018.]</ref name="Beer Temple interview with De Garde"></ref> (~14 minutes [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) or a bad barrel resulting in off woody flavor spontaneous fermentation. 2017.]</ref name="Spontaneous Sour Hour" /> . However, these reports are anecdotal based on visual fermentation and microbe analysis was not done in many cases. De Roos et al. (~1:31 2018) reported that for wort that is pre-acidified to a pH of 4.5, and after an initial drop in) or excessive O2 exposurepH to 3. In addition 8 by enterobacterial and acetic acid bacteria, the pH rose to 4.0 during the beer inside such barrels being dumpedsecondary fermentation phase, indicating that the yeast consumed some of the organic acids that were produced during the barrel itself is also often discarded initial enterobacteria phase <ref name="Beer Temple interview with De GardeRoos_2018_2"></ref> (~14 minutes in). Homebrewers who are fermenting spontaneously may expect that from time to time they will need to dump a batch.

===Microbial Succession During Fermentation=Third Stage: Acidification====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" />. This stage is dominated by lactic acid bacteria (LAB), primarily ''[[Pediococcus]]'' and sometimes ''[[Lactobacillus]]''. Several organic acids are produced during this stage with the majority of them being lactic acid and acetic acid, resulting in the pH of the beer dropping to below 3.5 <ref name="Van Oevelen et al., 1977" /><ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. Other sources describe the acidification and maturation phases 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" />. Specifically, the ratio of lactic acid to acetic acid greatly impacts the flavor profile of the beer. Lactic acid can range from 1.5 to 10 g/l, where as acetic acid is hopefully limited to 1.5 g/l due to he more harsh acidic flavor of acetic acid <ref name="Bongaerts_2021" />. 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" />.

The number of different species found in lambic and spontaneously fermented beers acidification phase is very large and diverse from brewery to brewery and batch to batch, however, scientific research in Belgium and also accompanied by the US has shown a regular general pattern to the microbial succession growth of spontaneous fermentation beer at the genus levelacetic acid bacteria (AAB), with only minor genera differences between Belgian lambic beers and American spontaneous ale. This difference which can be undesirable if this growth is attributed excessive since it leads to different microbes being present in different breweries <ref name="Van Oevelen et al., 1977">greater [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.Acetic Acid|acetic acid]</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 stageproduction (in high quantities, 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] acetic acid smells and [http://laboratoryresearch.blogspot.com/2008/07/yeasts-tastes like vinegar and-yeastlike-fungi.html?m=1 oxidative yeasts] that produce large amounts of DMS which can be smelled during is very harsh on the early stages of fermentation (see [[Dimethyl Sulfide]] for more detailspalate and throat)as well as acetoin. Although spontaneous ales have a common pattern of fermentation by groups of These microbes include species from the 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, ''KlebsiellaAcetobacter'' spp., and ''EnterobacterGluconobacter'' spp.,'' Pectobacterium carotovorum'', The species diversity of these genre is lower than during the primary stage due to acid and ''Serratia ureilytica'' have been foundethanol selecting for species that are more tolerant to these harsher conditions. In Belgian lambic producersFor example, ''Enterobacter'' sppDe Roos et al., such as (2018) reported high numbers of ''Enterobacter aerogenesAcetobacter pasteurianus'', ''Enterobacter cloacae'', ''Enterobacter hormaechei'' which contains extra genes that code for acid and ethanol tolerance more so than other species of ''Enterobacter kobeiAcetobacter'', ''Klebsiella aerogenes''in lambic from month 3 to month 6, with it disappearing around month 9-13 as ''Klebsiella oxytocaPediococcus damnosus'', took its place. ''Klebsiella varicolaAcetobacter lambici'', ''Escherichia coli'', ''Hafnia alvei'', ''Hafnia paralvei'', and ''Citrobacter freundii'', have been is another species found in lambic, with ''E. cloacae'' during this stage and ''K. aerogenes'' as is well adapted to the most frequently found oneslambic environment due to its ability to break down maltooligosaccharides (dextrins) via maltooligosyl trehalose synthase. Although these enterobacteria contribute little These microbes are dependent on oxygen in terms of gravity drop over the first month of fermentation order to metabolize ethanol into acetic acid (they mostly consume sucrose in the wortwith acetaldehyde produced as an intermediary step), they do contribute aroma and flavor compounds acetoin from lactic acid and precursors during are found on the initial stages surface of spontaneous fermentation, particularly acetoin, 2,3 butanediol, acetic acid, lactic acid, succinic acid, DMS, acetaldehyde, long-chain fatty acids the wort where oxygen is available. The beer/air interface (these play a role in both flavor impact and providing nutrients for yeast later in or surface of the beer that interfaces with the fermentation processair above it), and small amounts is also where higher concentrations of glycerolacetic acid, ethyl acetate, and higher alcohols which might form esters in acetoin are found due to the AAB being present there rather than deeper within the later stages of fermentation. Enterobacteria can also contribute beer (this is similar to the production of [https://en.wikipedia.org/wiki/Biogenic_amine biogenic amines[Flanders Red Ale]] 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., 1992Roos_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.ncbiaroxa.nlm.nih.govcom/pubmedbeer/30246252?dopt=Abstract Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production-flavour-standard/acetic-acid Aroxa website. Jonas De Roos and Luc De Vuyst"Acetic Acid". Retrieved 11/19/2018. DOI: 10.1002/jsfa.9291.]</ref>, although one study by Curtin et al. and the levels of acetic acid in Belgian gueuze/lambic being reported finding at least small populations in the range of enterobacteria as late as up to 4.5 months 727-2240 ppm, acetic acid levels in this range is an important flavor compound in spontaneously fermented beers <ref name="curtain_asbc_2018">[httpshttp://wwwbeachwoodbbq.asbcnet.orgcom/labpdf/webinars/webinars/Pages/funkyFermentationsWebinarBBAIBLTBLENDERY.aspx Chris Curtinpdf Ryan Fields. ASBC webinar: "Funky FermentationsBrewing Beer in America Inspired By the Belgian Lambic Tradition". 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 During a part second phase of growth of the core microbes acetic acid bacteria starting at week 7 in spontaneous fermentationlambic casks, it has been shown that ''Saccharomyces cerevisiae'' is metabolically stimulated significantly more acetoin (moldy/must flavor when co-fermented with some of these species, allowing the ''S. cerevisiae'' to consume more glucose and nitrogen and to more quickly replicate above 50 ppm <ref name="Roos_2018" />. De Roos et al (2018) reported significant populations of the enterobacteria species ''Klebseilla variicola'', ''Klebsiella oxytoca'', and the yeast species ''Hanseniasspora uvarum'', ''Saccharomyces cerevisiae'' during the first week or two of lambic fermentation that was pre-acidified (see [https://www.ncbimorebeer.nlm.nih.gov/pmccom/articles/PMC6252343Fatty_Flavors_Diacetyl "Fatty Flavors and Diacetyl - Should Your Beer Be Fat-Free?". MoreBeer website. Scott Bickham. Retrieved 04/figure04/F32023.]</?report=objectonly Figure 3]ref>). '''Wort or beer fermenting during this stage should not be consumed due to was found in the fact that some top portion of these are pathogenic the lambic casks above flavor threshold. Acetic acid bacteria has been shown to reduce lactic acid into acetoin, and pose potential health risksin another study by De Roose et.al (2023) the researchers found that ''Acetobacter lambici'' Oxidative yeasts are also present during in lambic samples that were tested had the first stage of fermentation, including species of ''Rhodotorula'', ''Candida'', ''Cryptococcus'', ''Hanseniaspora'', and ''Pichia'', some of which might survive pre-acidification genetic capability to utilize lactic acid as a food source <ref name="Bokulic et al., 2012Roosa_2024" />. 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 Acetoin was gradually reduced (using his wifepresumably metabolized by ''Brettanomyces''s Facebook account). Milk The Funk Facebook group post on analysis of spontaneous fermentation below flavor thresholds at 42 North Brewing Co. 11/09/2018.]month 9 until it reached near 0 ppm around month 18 <ref name="De_roos_AAB_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). ''Hanseniasspora 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=Fourth Stage://www.spontanmanc.co.uk/?pMaturation==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 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 ''[[SaccharomycesBrettanomyces]]'' dominated stage of fermentation is followed by prolonged and gradual acid and flavor development accompanied by the final points of attenuationfor more information), 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 as well as lactic acid bacteria (LAB), primarily from the genera ''[[Pediococcus]]'' and sometimes to a lesser extent ''[[Lactobacillus]]'', and a "maturation phase" driven by certain other yeast species. The most abundant species of ''[[Brettanomyces]]'' <ref name="Van Oevelen et alfound in spontaneously fermented beer are strains of ''B. bruxellensis'' (''B.lambicus'' is often found, 1977" />. Other sources describe these but has been reclassified as one extended maturation phase with acidification from a strain of ''PediococcusB. bruxellensis'' and ). ''BrettanomycesB. bruxellensis'' growth occurring simultaneously <ref name="Spitaels et alwas first isolated from English stock ales in 1904 and then first isolated from lambic in 1921 by Belgian researchers Kufferath and Van Laer., 2015" /><ref name="Bokulic et al''B., 2012" /><ref name="Spitaels et alanomalus'' and ''B.custersianus'' have also been found, 2014" />but to a lesser extent than ''B. When the wort is pre-acidified, the acidification and maturation phases overlap <ref name="Roos_2018" />bruxellensis''. Other yeasts such as ''CandidaPichia membranifaciens'', ''CryptococcusDebaryomyces hansenii'', and ''TorulopsisWickerhamomyces anomalus'' are examples of other yeast species that have also been isolated from mature found to a lesser extent in lambic, although their impact other than possibly being involved in during the formation of a pellicle is unknown maturation phase <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. name="Van Oevelen Met al. Spaepen P. Timmermans H. Verachtert. , 1977. DOI: https:" /><ref name="Roos_2018" /doi.org/10.1002/j.2050-0416.1977.tb03825.x.]><ref name="Roos_2018_2" /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., 1976Bongaerts_2021" /><ref name="Spaepen et al., 1978Roosa_2024" />. Homebrewer Caleb Buck reported data on several batches of homebrewed spontaneously fermented beer and observed a slower drop in gravity for In some batches than others over about but not all lambic, a 7 month period (see [http://wwwshift from ''B.archaicpursuitbruxellensis'' to ''B.com/2018/08/2017-coolship-experiment-hopping-rate.html?m=1 this graph 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 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) more aerobic environment <ref name="Roos_2018_2Bongaerts_2021" />.

The acidification This phase is also accompanied by the growth of acetic acid bacteria (AAB)generally begins somewhere around month four to eight, which can be undesirable if this growth is excessive since it leads to greater [[Acetic Acid|acetic acid]] production with these microbes completely dominating at around 9-13 months <ref name="Roos_2018_2" /><ref name="curtain_asbc_2018" />(~26 minutes in high quantities, acetic acid smells and tastes like vinegar and is ). Additional attenuation occurs very harsh on the palate and throat) as well as acetoinslowly for another 7-18 months. These microbes include species De Roos et al. (2018) reported a gradual drop from 4 Plato to 0.5 °Plato during the genera of ''Acetobacter'' and maturation phase. <ref name="Roos_2018_2" />. During this extended maturation phase, ''GluconobacterBrettanomyces''. These microbes are dependent on oxygen in order continues to metabolize ethanol into acetic acid (with acetaldehyde produced as an intermediary step) and acetoin from lactic acid and are found on ferment the surface of residual sugars leftover in the wort where oxygen is available. The beer/air interface (or surface using intra- and extracellular alpha-glucosidase, and produces most of the beer that interfaces with final aromatic and flavor compounds in the air above it) is also where higher concentrations form of acetic acid esters, phenols, and acetoin are fatty acids found due to the AAB being present there rather than deeper within the beer in finished Belgian lambic and other spontaneously fermented beers (this is similar to see [[Flanders Red AleBrettanomyces#Brettanomyces_Metabolism|''Brettanomyces'' metabolism]]). It has been shown that During the maturation phase, a [[pellicle]] is formed from the ''Brettanomyces'', as well as oxidative yeasts from the species of AAB found in lambic genera ''Pichia'', ''Candida'', ''Cryptococcus'', and American spontaneous ales have adapted to high concentrations of ethanol and acetic acid ''Torulspsis'' <ref name="Van Oevelen et al., 1977" /><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. name="Acetic AcidRoos_2018_2". Retrieved 11/19/2018.]><ref name="Bongaerts_2021" /ref>, . It is thought that the pellicle and the levels presence of acetic acid in Belgian gueuze/lambic being reported these oxidative yeasts might reduce oxygen influx, and thus assist in inhibiting the range growth of 727-2240 ppm, acetic acid levels in this range is an important flavor compound in spontaneously fermented beers bacteria <ref>[httphttps://beachwoodbbqpdfs.comsemanticscholar.org/pdf8c12/BBAIBLTBLENDERY9985b9f1264179fe2e2f779bae1ff3e51a54.pdf Ryan FieldsJacques De Keersmaecker. "Brewing The Mystery of Lambic Beer in America Inspired By the Belgian Lambic Tradition". 2018Scientific American, Inc. 1996.]</ref><ref name="Spitaels et al., 2015" />. De Roos et al. (2018) reported high numbers of ''Acetobacter pasteurianus'' from month 3 to month 6however, 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 this has not been proven in a scientific manner 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 we know of fermentation <ref name="curtain_asbc_2018" />(~26 minutes in).

The fourth and last phase of spontaneous fermentation, also known as During the extended maturation phase, is dominated by ''[[Brettanomyces]]'' yeast, which is a genus of yeasts that are highly tolerant of low pHbeer may become "sick" or "ropey", 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 though not all producers get this <ref name="Roos_2018_2Spontaneous Sour Hour" />(~1:10 min in) <ref name="curtain_asbc_2018Vinnie sour beer talk" >[http://www.thebrewingnetwork.com/post1863/Recording of Vinnie's talk at NHC]</ref>(~26 minutes 1:44 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_2Vinnie on the Session Jan 2010" />(~3:44 in). During this extended maturation phaseThis is the result of exopolysaccharides, which some ''BrettanomycesPediococcus'' continues strains are known to ferment the residual sugars left over produce. These exopolysaccharides can be broken down by other microbes present in the beer using intra- and extracellular alpha-glucosidase, and produces most of relieving the final aromatic and flavor compounds in the form beer of esters, phenols, and fatty acids found in finished Belgian lambic and other spontaneously fermented beers its "sickness" (see [[Brettanomyces#Brettanomyces_Metabolism|this exopolysaccharide breakdown is generally attributed to ''Brettanomyces'' metabolism]]). The most abundant species of ''Brettanomyces'' found Beer may also become "sick" in spontaneously fermented beer are strains of ''Bthe bottle during bottle conditioning. bruxellensis'' (''B. lambicus'' This is often found, but has been reclassified as a strain of likely due to enhanced ''B. bruxellensisPediococcus''). During the maturation phase, a [[pellicle]] is formed activity from the ''Brettanomyces''additional fermentable sugar, as well as oxidative yeasts from in the genera ''Pichia'', ''Candida'', ''Cryptococcus'', and ''Torulspsis'' form of simple sugars or beer which has not completely attenuated yet <ref name="Van Oevelen et al., 1977Vinnie sour beer talk" /><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>[httpshttp://pdfswww.semanticscholarthebrewingnetwork.orgcom/8c12post1863/9985b9f1264179fe2e2f779bae1ff3e51a54.pdf Jacques De Keersmaecker. "The Mystery Recording of Lambic Beer". Scientific American, Inc. 1996.Vinnie's talk at NHC]</ref>, however, this has not been proven (~1:47 in). A beer that is sick in the bottle will generally clear through the same process as a scientific manner that we know ofyounger aging beer when given appropriate time. See the [[Pediococcus#.22Ropy.22_or_.22Sick.22_Beer|Pediococcus]] page for more information.

During Acetic acid bacteria tend to be absent from bottled gueuze due to the extended maturation phase, a beer may become "sick" or "ropey", though not all producers get this <ref name="Spontaneous Sour Hour" /> (~1:10 min in) <ref name="Vinnie sour beer talk">[http://wwwanaerobic environment.thebrewingnetwork.com/post1863/ Recording of Vinnie's talk at NHC]</ref> (~1:44 in) <ref name="Vinnie on the Session Jan 2010"/> (~3:44 in). This is the result of exopolysaccharides, which some 'Brettanomyces'' and ''Pediococcusdamnosus'' strains are known to produce. These exopolysaccharides can be broken down by other microbes present in the beer relieving the beer often recoverable from bottles of its "sickness" (this exopolysaccharide breakdown is generally attributed to gueuze as old as five years, but ''BrettanomycesPichia membranifaciens''). A beer may also become "sick" in the bottle during bottle conditioning. This is likely due to enhanced and ''PediococcusSaccharomyces cerevisiae'' activity have also been recovered from additional fermentable sugartwo year old bottles of gueuze. After five years of storage, lactic acid bacteria was no longer found in the form of simple sugars or beer which has not completely attenuated yet bottled gueuze <ref name="Vinnie sour beer talkBongaerts_2021">[http://www.thebrewingnetwork.com/post1863/ Recording of Vinnie's talk at NHC]</ref> (~1:47 in). A beer which is sick in the bottle will generally clear through the same process as a younger aging beer when given appropriate time. See the [[Pediococcus#.22Ropy.22_or_.22Sick.22_Beer|Pediococcus]] page for more information.

As with regular beer, spontaneously fermented beer is made up of hundreds of different compounds that can contribute to the flavor and aroma. Not all compounds are perceived equally; some compounds can be predominantly tasted in the beer, while other compounds can have a synergistic or antagonistic effect with some compounds enhancing background notes and others providing key flavors and aromas. The highest concentration of a particular compound does not mean that it will be perceived the most since some compounds with low concentrations can be perceived very easily at low concentrations <ref name="witrick_2017">[https://www.mdpi.com/2306-5710/3/4/51 Thompson Witrick, K.; Duncan, S.E.; Hurley, K.E.; O’Keefe, S.F. Acid and Volatiles of Commercially-Available Lambic Beers. Beverages 2017, 3, 51. DOI: https://doi.org/10.3390/beverages3040051.]</ref>. Spontaneously fermented beers are usually characterized by the compounds produced by the complex and highly variable fermentation profile explained above. This generally includes the production of [[Lactic Acid|lactic acid]] by lactic acid bacteria and [[Acetic Acid|acetic acid]] by acetic acid bacteria and ''Brettanomyces'', which lend a sour and fruity flavor to the beer. ''Brettanomyces'' is responsible for many of the volatile aroma compounds, including [[Tetrahydropyridine|tetrahydropyridine]] (also produced by lactic acid bacteria), phenols such as 4-ethylguaiacol (smokey, spicy, clove) and 4-ethylphenol (barnyard, horsey, spicy, smokey, medicinal, Band-Aid), and esters such as ethyl acetate (pineapple in low concentrations, nail polish in high concentrations), ethyl lactate (fruity, creamy, rum-like), ethyl caproate (fruity/aniseed), ethyl caprylate (fruity with creamy mushroom and cognac notes), and phenylethyl acetate (sweet honey and rose-like). ''Brettanomyces'' can also produce volatile fatty acids such as [[Isovaleric Acid|isovaleric acid]] <ref name="Roos_2018" /><ref name="Roos_2018_2" />. Some strains of ''Brettanomyces'' are also known to release the enzyme beta-glucosidase to break down glycosides, which can result in the release of flavor compounds. This enzyme is also responsible for allow ''Brettanomyces'' to consume cellobiose (wood from barrels). However, Daenen et al. (2007) found that none of the ''B. bruxellensis'' strains isolated from lambic could utilize cellobiose, but strains of ''B. anomalus'' and ''B. custersianus'' isolated from lambic could utilize cellobiose, indicating that not all ''Brettanomyces'' strains (especially ''B. bruxellensis'') can break down glycosides or use cellobiose in wood as a food source (see [[Brettanomyces#Glycosides_and_Beta-Glucosidase_Activity|beta-glucosidase activity]] for more information). See the [[Brettanomyces#Secondary_Metabolites|''Brettanomyces'' secondary metabolites]] page for a complete list of flavor compounds that ''Brettanomyces'' can produce. Other intermediate flavor and aroma compounds are creating during the fermentation process. For example, De Roos et al. (2018) reported the production of acetoin from week 3 to month 6 in lambic beers, and then a gradual decline after that. This corresponded with the growth of acetic acid bacteria (AAB), and the production of acetoin was attributed to the AAB oxidizing lactic acid. 2,3-Butanediol and 2,3-butanedione (diacetyl) were not found during the entire age of the lambics studied, so the full conversion of acetoin to these compounds never occurred. The decline of acetoin during the maturation phase was attributed to ''Brettanomyces'', which is known to occur when oxygen is limited. Malic acid was depleted as the lactic acid bacteria started to grow from month 6-9, and lactic acid increased (both D-lactic acid and L-lactic acid in nearly equal amounts at around 2.0 g/l), indicating malolactic fermentation occurred during this time. After the acidification phase and during the maturation phase where ''Brettanomyces'' and ''Pichia membranifaciens'' were dominant, small amounts of malic acid were produced, suggesting that maybe these yeasts are capable of producing small amounts of malic acid (less than 20 mg/l of malic acid was in the lambic beer at 24 months). Gluconic acid and citric acid, which were presumably introduced from the brewing process, were present at fairly stable levels during the entire fermentation process, with gluconic acid seeing a slight spike during the growth of acetic acid bacteria (~50 mg/l and ~220 mg/l in the final beers) <ref name="Roos_2018_2" />. As bottles of packaged beer age, they tend to increase in lactic acid and ethyl lactate concentrations as years pass, which also corresponds with a slow decrease in dextrins <ref name="Spitaels_2015_Bottles">[https://www.sciencedirect.com/science/article/pii/S0740002014002548 Microbiota and metabolites of aged bottled gueuze beers converge to the same composition. Freek Spitaels, Simon Van Kerrebroeck, Anneleen D. Wieme, Isabel Snauwaert, Maarten Aerts, Anita Van Landschoot, Luc De Vuyst, Peter Vandamme. 2015.]</ref>.

Other intermediate flavor and aroma compounds are creating during Below is a collection of data on spontaneously fermented beer sampled from bottles available in the market; see the fermentation processdata sources for standard deviations, methodology, etc.; a dash (-) means not reported or measured: {| class="wikitable sortable"|-! Brand !! pH !! Total Acidity (g/L) !! Lactic Acid (mg/L) !! Acetic Acid (mg/L) !! Data Source|-| Hanssens Artisan Gueuze (vintage not reported) || 3.23 || 7.83 || 1389 || 1642 || Witrick et al. (2017) <ref name="witrick_2017" />|-| 3 Fonteinen Gueuze (vintage not reported) || 3.24 || 5.71 || 1294 || 1204 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Oude Gueuze Boon (vintage not reported) || 3.43 || 2.62 || 1228 || 723 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Gueuze Boon (vintage not reported) For example, De Roos || 3.52 || 2.71 || 995 || 1137 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Oude Gueuze Vielle (vintage not reported) || 3.44 || 2.74 || 1094 || 1019 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Girardin Gueuze (vintage not reported) || 3.44 || 4.96 || 1403 || 1499 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Lindemans Cuvee Renée (vintage not reported) || 3.62 || - || 2557 || 916 || Witrick et al. (20182017) <ref name="witrick_2017" />|-| Cantillon Gueuze (vintage not reported the production of acetoin from week ) || 3.44 || 3.29 || 1417 || 1224|| Witrick et al. (2017) <ref name="witrick_2017" />|-| Cantillon Bio (vintage not reported) || 3 to month 6 in lambic beers.53 || 4.42 || 1658 || 1473 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Cantillon Gueuze; Bottled 2013, Bottle 1 || - || - || 4500 || 1360 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2013, and then a gradual decline after thatBottle 2 || - || - || 3800 || 1050 || Spitaels et al. This corresponded with the growth of acetic acid bacteria (AAB2015)<ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2013, and the production of acetoin was attributed to the AAB oxidizing lactic acidBottle 3 || - || - || 6200 || 2150 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2011, Bottle 1 || - || - || 6200 || 1890 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2011, Bottle 2|| - || - || 5700 || 1880 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2011,Bottle 3|| - || - || 5300 || 1820 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, Bottle 1 || - || - || 7500 || 1790 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-Butanediol and | Cantillon Gueuze; Bottled 2010, Bottle 2|| - || - || 8500 || 2020 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010,Bottle 3|| -butanedione || - || 10600 || 2240 || Spitaels et al. (diacetyl2015) were not found during the entire age of the lambics studied<ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, Bottle 1 || - || - || 9600 || 1220 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, so the full conversion of acetoin to these compounds never occurredBottle 2 || - || - || 13800 || 1270 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, Bottle 3 || - || - || 11900 The decline of acetoin during the maturation phase was attributed to ''Brettanomyces''|| 1290 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, Bottle 1 || - || - || 13300 || 1850 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, Bottle 2 || - || - || 17600 || 1800 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, Bottle 3 || - || - || 11300 || 1430 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, Bottle 1 || - || - || 10900 || 720 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, Bottle 2 || - || - || 14100 || 930 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, which is known to occur when oxygen is limitedBottle 3 || - || - || 10200 || 710 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-|} Reported volatile compounds in mg/L; a dash (-) means not reported or measured: {| class="wikitable sortable"|-! Brand !! Isovaleric Acid !! Ethyl Octanoate !! 4-Ethylphenol !! 4-Ethylguaiacol !! Octanol !! Ethyl Decanoate !! Ethyl Undecanoate !! Ethyl Lactate !! Ethyl Acetate !! Data Source |-| Hanssens Artisan Gueuze (vintage not reported) || - || 2.74 || 0.57 || 1.36 || 0.084 || - || - || - || 66.9 || Witrick et al. (2017) <ref name="witrick_2017" />|-| 3 Fonteinen Gueuze (vintage not reported) || - || 1.36 || 0.44 || 0.99 || 0.034 || - || - || - || 22.1 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Oude Gueuze Boon (vintage not reported) || 2.3 || 1.66 || 0.28 || 0.52 || 0.02 || - || - || - || 17 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Gueuze Boon (vintage not reported) || - || 1.62 || 0.32 || 0.97 || 0.02 || - || - || - || 11.8 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Oude Gueuze Vielle (vintage not reported) || 1.92 || 2.68 || 0.57 || 1.06 || 0.031 || - || 16.3 || - || 22.3 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Girardin Gueuze (vintage not reported) || 2.95 || 2.22 || 0.28 || 0.52 || 0.02 || - || - || - || 21.4 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Lindemans Cuvee Renée (vintage not reported) || 3.01 || 5.67 || 1.13 || 5.77 || 0.041 || - || 46 || - || Not detected || Witrick et al. (2017) <ref name="witrick_2017" />|-| Cantillon Gueuze (vintage not reported) || 2.15 || 5.17 || 1.08 || 2.44 || 0.052 || - || 28.8 || - || 28.4 || Witrick et al. (2017) <ref name="witrick_2017" />|-| Cantillon Bio (vintage not reported) || 2.94 || 4.52 || 0.96 || 2.1 || 0.025 || - || 8. Malic acid was depleted as the lactic acid bacteria started to grow from month 6|| -|| 46.9|| Witrick et al. (2017) <ref name="witrick_2017" />|-| Cantillon Gueuze; Bottled 2013, Bottle 1 || - || - || - || - || - || 4.6 || - || 68 || 118 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2013, Bottle 2 || - || - || - || - || - || 11.8 || - || 44 || 163 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2013, and lactic acid increased Bottle 3 || - || - || - || - || - || 11.3 || - || 195 || 130 || Spitaels et al. (both D2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2011, Bottle 1 || - || - || - || - || - || 3.3 || -lactic acid and L|| 166 || 170 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-lactic acid in nearly equal amounts at around | Cantillon Gueuze; Bottled 2011, Bottle 2|| - || - || - || - || - || 8.7 || - || 175 || 252 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2011, Bottle 3 || - || - || - || - || - || 7.5 || - || 162 || 292 || Spitaels et al.0 g(2015) <ref name="Spitaels_2015_Bottles" /l>|-| Cantillon Gueuze; Bottled 2010, Bottle 1 || - || - || - || - || - || 2.9 || - || 249 || 203 || Spitaels et al. (2015)<ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, indicating malolactic fermentation occurred during this timeBottle 2 || - || - || - || - || - || 8.2 || - || 295 || 283 || Spitaels et al. After the acidification phase and during the maturation phase where ''Brettanomyces'' and ''Pichia membranifaciens'' were dominant(2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, small amounts of malic acid were producedBottle 3 || - || - || - || - || - || 6.5 || - || 332 || 285 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, indicating that these yeasts are capable of producing small amounts of malic acid Bottle 1 || - || - || - || - || - || 2.2 || - || 371 || 125 || Spitaels et al. (less than 20 mg2015) <ref name="Spitaels_2015_Bottles" /l of malic acid was in the lambic beer at 24 months>|-| Cantillon Gueuze; Bottled 2008, Bottle 2 || - || - || - || - || - || 5.7 || - || 413 || 319 || Spitaels et al. (2015)<ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, Bottle 3 || - || - || - || - || - || 5.6 || - || 415 || 246 || Spitaels et al. Gluconic acid and citric acid(2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, which were presumably introduced from the brewing processBottle 1 || - || - || - || - || - || 2.0 || - || 630 || 140 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, were present at fairly stable levels during the entire fermentation processBottle 2 || - || - || - || - || - || 3.4 || - || 592 || 197 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, with gluconic acid seeing a slight spike during the growth of acetic acid bacteria Bottle 3 || - || - || - || - || - || 2.7 || - || 464 || 178 || Spitaels et al. (~50 mg2015) <ref name="Spitaels_2015_Bottles" /l and ~220 mg>|-| Cantillon Gueuze; Bottled 1996, Bottle 1 || - || - || - || - || - || 1.8 || - || 526 || 89 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" /l in the final beers>|-| Cantillon Gueuze; Bottled 1996, Bottle 2 || - || - || - || - || - || 3.9 || - || 778 || 150 || Spitaels et al. (2015) <ref name="Roos_2018_2Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, Bottle 3 || - || - || - || - || - || 2.5 || - || 556 || 169 || Spitaels et al.(2015) <ref name="Spitaels_2015_Bottles" />|-|}

[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>.  Some government regulations and rules exist for upper limits of hystamine in meat and fish (although these limits could be applied to other food products). For more informationexample, see histamine levels in meat and fish must be less than 50 mg/kg in the US and less than 200 mg/kg in the UK <ref>[httphttps://suigenerisbrewingwww.mdpi.com/index2304-8158/8/2/62/htm Impact of Biogenic Amines on Food Quality and Safety. Claudia Ruiz-Capillas and Ana M. Herrero. 2019. DOI: https://doi.org/10.3390/foods8020062.]</ref>.php Some countries have set an upper limit of histamine in wine to be anywhere from 2-10 mg/2019l <ref>[https://ifst.onlinelibrary.wiley.com/doi/abs/10.1111/ijfs.12833 Biogenic amines in wine: a review. Yan‐Yun Guo, Yan‐Ping Yang, Qian Peng, Ye Han. 2015.]</01ref>. Loret et al. (2005) quotes the upper limit of individual amines to generally be safe for consumption at below 10 mg/22l <ref name="loret_2005" />.  Loret et al (2005) examined the levels of biogenicamines 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 aminesabove 10 mg/ l <ref name="Fact loret_2005" />. Another study in Portugal measured biogenic amines in 5 Portuguese craft beers (4 ales and 1 lager; no mixed fermentation or Fiction – 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 BeerCraft 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 Drthe same group of researchers in 2024 confirmed that ''P. Bryan Heit]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"/>.

Biogenic The biogenic amines in content of spontaneously fermented beers are produced mostly by enterobacteria, but lactic acid bacteria and yeasts can also produce them. Wort beer that is pre-not acidified greatly reduces the production before fermentation is not likely to be high enough to cause serious physical harm. Large amounts of biogenic amines, but small levels can still beer must be foundconsumed before advisable limits are reached. These levels are below However, the levels found in cheese and fermented sausage and are well below the levels that are acceptable for additive effect of different amines consumed with a large amount of beer might cause health (histamine levels less than 50 mg/kg related reactions in the US and less than 400 mg/kg in the UK; these levels are usually regulated for meets and fish<ref>some people. See [httpshttp://www.mdpisuigenerisbrewing.com/2304-8158index.php/8/2/62/htm Impact of Biogenic Amines on Food Quality and Safety. Claudia Ruiz-Capillas and Ana M. Herrero. 2019. DOI: https:/01/doi.org22/10.3390/foods8020062.]</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 / "Fact or Fiction – Biogenic Amines in a coolship and spontaneously fermentedBeer" by Dr. They found that the initial wort had low concentrations Bryan Heit; an analysis 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, spontaneously fermented beer 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" />associated health concerns] for more information.

===Notes on the Source of Microbes===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 is often claimed 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. 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 samples the interior of barrels and foeders used Some yeast species that have been found in limited capacity in one lambic brewery that uses high-pressurized hot water and sulfur dioxide to clean them, and found surviving colonies of such as ''Brettanomyces Wickerhamomyces 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 barrelshave been isolated from the air of coolship rooms, but not others. They they have also found high levels been isolated from the air of ''Cellulosimicrobium'' and ''Acinetobacter''cellar rooms, which indicating that they could have not been found introduced to be important for lambic fermentation; the authors suspected that these were living wort/beer in the wood rather than in the beer since they can metabolize cellulosecellar as well. Although they Additionally, yeast and bacteria species that are probably also not important to the most abundant during lambic fermentation of lambic, molds were found in the more porous barrels before cleaning, including (''AspergillusSaccharomyces'' and , ''PenicilliumBrettanomyces''. 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 ''SaccharomycesPediococcus'' , and ''Pichia'' acetic acid bacteria) have not been isolated from the air of coolship rooms <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: 10.1128name="Bongaerts_2021" /AEM.02226-18.]</ref>.

Professor Chris Curtin from Oregon State University presented 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 results interior of a study by his team where 50 barrels of three different vintages of spontaneously fermented, and foeders used in one lambicbrewery that uses high-inspired beer was sampled three times over a 9 month period. These 50 barrels represented three batches of the same recipe brewed in 2013, 2015pressurized hot water and sulfur dioxide to clean them, and 2016. With the exception found surviving colonies of a small number of barrels''Brettanomyces anomalus'', they all contained the same or similar ''Brettanomyces B. bruxellensis'' yeast, ''B. There was no significance between the three different batches. Two of the 2015 barrels had custersianus'', ''Pediococcus'', and to a different species of lesser extent ''BrettanomycesAcetobacter'' (and ''clausseniiLactobacillus''). Two of the 2016 barrels had more The ''Saccharomyces cerevisiaeAcetobacter''. This study indicated might be linked to older barrels that barrel to barrel variation as far as yeast goes is fairly smallare more porous. Bacteria populations clustered much closer based on the vintage (but not the barrel). The 2013 batch was dominated by ''GluconobacterPichia'', ''Debaryomyces hansenii'' and ''AcetobacterCandida''were also found in abundance in some barrels, while the 2015 batch was dominated only by but not others. They also found high levels of ''AcetobacterCellulosimicrobium'', and the 2016 batch was dominated by ''LactobacillusAcinetobacter''. This indicated , which have not been found to be important for lambic fermentation; the authors suspected that these were living in the vintage of the batch plays a major role wood rather than in determining which bacteria will be the dominant bacteria during maturation, but individual barrels generally do beer since they can metabolize cellulose. Although they are probably also not (although a small number of barrels matured faster than important to the majority fermentation of lambic, molds were found in the more porous barrels before cleaning, including ''Aspergillus'' and were dominated by bacteria that represented the more mature vintage)''Penicillium''. Despite No molds survived the sulfuring process and the lack diversity of major variation between microbes was far higher in barrels, Curtin determined that some barrels can introduce microbial variationthan it was for foeders, perhaps due to insects transferring microbes or differences in because of the higher level of oxygen ingress between different exposure in barrelscompared to foeders, and the ability although foeders had a much higher than expected amount of yeast surviving ''Saccharomyces'' and bacteria to live within the inner surface ''Pichia'' <ref>[https://aem.asm.org/content/early/2018/10/15/AEM.02226-18 The interior surfaces of the wooden barrels and potentially survive cleaning procedures 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 name="curtain_asbc_2018" /> (~29 minutes in).

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" />.  Landschoot et al. (2015) attempted to recover yeasts and bacteria sampled from the brewery walls, ceilings, and coolship in an industrial brewery in West-Flanders that also produces lambic. They were unable to 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 ''Klebsiella oxytoca'', ''Bacillus'', and ''Staphylococcus'', 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 3 weeks. ''S. cerevisiae'', ''S. pastorianus'', ''D. bruxellensis'', ''P. damnosus'', and a diverse range of acetic acid bacteria were the dominate species of microbes found during the fermentation of lambic in this brewery. After boiling, the brewery cooled its wort to 40°C using an industrial heat exchanger before pumping it into the coolship to reside overnight for 24 hours. The foeders in this brewery are cleaned with a pressure washer and are no attempt was made to pasteurize or sanitize the foeders with heat or chemicals. The inside surfaces of the foeders were sampled and these samples were the only samples that contained microbe species that were found during the fermentation of the lambic. The authors concluded that the microbes fermenting lambic at this brewery are introduced into the wort via the wooden inner surfaces of the foeders <ref name="Landschoot_2015"/>.  De Roos et al. (2024) reported finding a very diverse set of species in wort sampled from coolships after cooling (100-200 mL samples). Using DNA sequencing techniques and in order of abundance, they found ''H. uvarum'', ''S. cerevisiae'', ''Penicillium roqueforti'', ''Acinetobacter guillouiae'', ''Lactococcus raffinolactis'', ''Geotrichum candidum'', ''Chryseobacterium bovis'', ''Flavobacterium hibernum'', ''Yarrowia lipolytica'', and ''Trichococcus flocculiformis''. However, the only species from the coolship samples that were found during fermentation in two Italian wine barrels were ''H. uvarum'' and ''S. cerevisiae''. The authors concluded that the wooden casks themselves, despite heavy cleaning methods, contributed the majority of impactful microbe species during fermentation and aging, including ''Brettanomyces'' <ref name="Roosa_2024"/>. The authors of the study write (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 in the spontaneous inoculation of the fermenting wort and maturing beer during lambic beer production. Although shotgun metagenomics revealed that the cooled wort had the highest diversity and evenness, its harboring species did not contribute to the wort fermentation and beer maturation in the casks.'' <ref name="Roosa_2024"/></blockquote> Various brewer anecdotes from experiments appear to offer 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.

It is worth noting that it appears to be possible for spontaneous fermentations to not harbor lactic acid bacteria or ''Brettanomyces''. For example, Andy Mitchell from New Belgium Brewing had a homebrewed spontaneous fermentation analyzed by New Belgium's lab after a year of age, and they only found a diastatic strain of ''Saccharomyces cerevisiae'' var ''diastaticus'' living in the beer and no lactic acid bacteria or ''Brettanomyces'' (diastatic strains of ''S. cerevisiae'' are generally not found in the wild and this could have come from the barrel from previous inoculations; however, the barrel was stored for 1.5 years prior to this being used with water, potassium metabisulfite, and citric acid). Andy reported that the beer was also not sour and did not have any a ''Brettanomyces'' character flavor profile <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1879283728766499/ Andy Mitchell. Milk The Funk thread regarding homebrewed batch of spontaneously fermented beer with no ''Brettanomyces'' or LAB. 11/10/2017.]</ref>. ===Blending (and Dumping)===[[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.