Changes

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

==Defining ''Spontaneous Fermentation''==In the most romanticized view of spontaneous fermentation, the microbes which inoculate the wort in the coolship are sourced exclusively from the ambient environment outside the brewery. Scientific publications have suggested that in the case of some producers, these microbes may be resident in the brewhouse <ref name="Bokulic et al., 2012">[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035507/ Bokulich et al, 2012]</ref>. This is supported by the reluctance of lambic brewers to alter their facilities (remodeling, moving, painting, etc.) and the spraying of lambic on the walls of new buildings <ref>[https://www.facebook.com/permalink.php?story_fbid=888263374558973&id=110627652322553/ Cantillon Facebook post 5-February-2015]</ref> <ref>[http://www.latisimports.com/assets/uploads/2009/11/MBA_Boon_October_Article.pdf/ Modern Brewery Age Weekly 23-October-2009 Article by Peter Reid with Frank Boon, accessed 7-May-2015]</ref>. The microbes responsible for spontaneous fermentation may also be derived from the oak wooden [[Barrel|barrels ]] and/or foedres foudres which are often used to hold the fermenting beer, especially if the barrels/foudres have not been thoroughly cleaned <ref name="Spitaels et al., 2015">[http://www.sciencedirect.com/science/article/pii/S074000201500012X/ The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer. Spitaels et al., 2015]</ref>. Many Belgian lambic producers thoroughly clean their barrels using hot water/steam, mechanical agitation (--add such as is seen at [[Cantillon ref--]]), and/or burning sulfur <ref> Conversation between Dave Janssen and Steven Sonck of [[De Cam]], winter 2014</ref>; however even the most rigorous cleaning likely does not fully sterilize the barrels. In the case of lambic brewers , the microbes resident in barrels are spontaneous in origin, having been derived from years to decades of use in the brewery without any exposure to pitched cultures and the barrels may serve as a concentrating mechanism for the desired cultures. The role of barrels as an inoculating vessel is unclear as some producers report achieving excellent results in barrels which are new to the brewery and which are microbially clean (<ref name="Spontaneous Sour Hour" /> (~35 min in).We do not regard the use of well-cleaned barrels but still containing native microbes from previous use to invalidate spontaneous fermentation

A spontaneous native wild-microbe fermentation may also be achieved by ambiently inoculating small amounts of wort and growing up the spontaneously inoculated caught microbes to check for suitability. This process has often been called a "spontaneous starter" and is common in homebrew production <ref> [http://www.themadfermentationist.com/2011/04/ambient-spontaneous-yeast-starters.html The Mad Fermentationist Spontaneous Starters, accessed 7-May-2015]</ref> and , however, it is arguably more accurately described as one of the few methods of [http://www.milkthefunk.com/wiki/Wild_Yeast_Isolation bioprospecting]. Bioprospecting from ambient collection allows for the screening of the microbes to remove wild cultures with aggressive off -flavors and/or mold. This is not unlike the potential of used oak barrels, where well -performing barrels may be kept and used to inoculate ferment subsequent batches (where the organisms residing in the barrel can exert their influence on the batch) while poorly performing barrels may be discarded and removed from the brewery. This process does differ from oak barrels in that native microbes are cultured and pitched into the wort, rather than the additional inoculation being a result of porous surfaces that have not been fully sanitized. As different microbes survive and thrive in different environments, barrels or pre-screened and grown starters will probably not provide a complete profile of the microbes present in traditional spontaneous fermentation beers. However , a combination of a coolship to inoculate the wort with ambient/brewhouse resident microbes combined with a form of pre-screening such as barrel re-use and/or spontaneous starters may provide the full microbiota present in traditional spontaneously fermented products. For the purposes of this page, beers receiving additions of isolated cultures or bottle dregs are not treated as spontaneous and are discussed under [[Mixed Fermentation|mixed-culture fermentation]].

==Brewing Methods==There is some debate as to whether using spontaneous starters should still be termed "spontaneous fermentation" <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1225800400781505/ MTF Facebook thread about naming]</ref>. Each brewer will have to decide for him or herself what terms to use. We recommend transparency and clarity in descriptions and process to avoid potential confusion. In a stricter sense, beers using active inoculation with spontaneous starters might better be described with terms like 'wild beer', 'beer fermented with native microbes', or a description of the spontaneous starter process rather than entirely "spontaneous beers". Especially if the full wort volume was not ambiently cooled and the starter was allowed to grow for some time before mixing it in. An alternative term for the process itself includes something like "ambient bioprospecting". See [[Spontaneous_Fermentation#Alternative_Applications_of_Spontaneous_Fermentation|alternative applications of spontaneous fermentation]] below. Beers receiving additions of isolated cultures or bottle dregs are not spontaneous and are discussed under [[Mixed Fermentation|mixed-culture fermentation]] and [[Commercial_Sour_Beer_Inoculation|commercial sour beer inoculation]]. Ambient bioprospecting (or "spontaneous starters") are discussed in progress)more detail on the [[Wild_Yeast_Isolation#Catching|Wild Yeast Isolation]] page.

The traditional production It has been suggested by some that the terminology of "spontaneous fermentation" is not precise enough for describing the process. Other phrases such as "open-air inoculation", "non-selected fermentation", "ambient inoculation", "coolship ales", and "wild ales" are phrases that some people suggest for alternative nomenclature for spontaneously fermented beer employs a few main processes . However, the phrase "spontaneous fermentation" has been widely used by the Belgian lambic brewers to designate this process and goals including the production of a dextrinous wort, high hopping rates (usually with aged hops), term has been applied in the [https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A52016XC0514%2804%29 EU's TSG law for lambic]. This term has also been widely adopted by the American brewing industry to the point where many brewers and inoculation consumers alike recognize the meaning of the wort with a phrase "spontaneous fermentation" <ref>[[coolshiphttps://www.facebook.com/groups/MilkTheFunk/permalink/2056705564357647/?comment_id=2057416344286569&comment_tracking=%7B%22tn%22%3A%22R%22%7D Various MTF members. Milk The Funk Facebook group thread on the wording of 'spontaneous fermentation'. 04/12/2018.]]. Not all breweries producing spontaneously fermented beer employ these three techniques, but they are generally common among producers</ref>.

<gallery>File:Spontaneous starters 2.jpeg|Bioprospecting with an ambient starter File:DSCN2215.JPG|Bioprospecting with an ambient starterFile:Spontaneous starters 3.jpeg|Bioprospecting with an ambient starter</gallery> ==Wort Production== The traditional production of spontaneously fermented beer employs a few main processes and goals including the production of a dextrinous wort, high hopping rates (usually with aged hops), and inoculation of the wort by open cooling in a [[coolship]]. Not all breweries producing spontaneously fermented beer employ these three techniques, but they are generally common among producers. ===Mashing=== 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>. For more information on turbid mashing, see the [[Turbid Mash|Turbid Mashing]] page. ===Hopping=== Traditional Belgian lambic brewers use high hopping rates of aged hops in a long boil (~4 hours or more, which is due to the large volume of sparge water that is used during the [[Turbid Mash|turbid mashing]] process), however, American brewers making spontaneously fermented beer sometimes use a combination of aged and fresh dried hops. Historically, there is [http://www.horscategoriebrewing.com/2016/04/hops-in-spontaneous-fermentation.html some evidence] that lambic brewers used a combination of aged hops and fresh dried hops as well. The high hopping rates help to regulate [[Lactobacillus#Hop_Tolerance|bacterial activity]] and select for the desired bacteria (''[[Pediococcus]]'' rather than ''[[Lactobacillus]]''). Aging of the hops lowers the flavor/aroma impact the hops provide and also lowers the bitterness. The aged hops still do provide some bitterness as both oxidized alpha acids and oxidized beta acids can contribute to perceived bitterness and measured IBUs <ref name="OSU talk at CBC 2015"> Understanding How to Control Flavor and Aroma Consistency in Dry Hopped Beer. Dan Vollmer, Dan Sharp, Dr. Tom Shellhammer (Oregon State University). Oral presentation at the 2015 Craft Brewers Conference</ref>. Cantillon uses hops that are on average 2-3 years old at hopping rate of 250-300g/100 L (0.334-0.40 oz/gal) <ref name="Spontaneous Sour Hour">[http://www.thebrewingnetwork.com/membersarchive/sourhour2015_05_wildfriendship.mp3 The Sour Hour Episode 11 with Rob Tod and Jason Perkins from Allagash, Jean Van Roy from Cantillon, and Vinnie Cilurzo from Russian River]</ref>(~49 minutes in). See also the note about Cantillon's hopping on the [[Cantillon]] wiki page, as actual hopping rates may be slightly higher than the 250-300 g/100 L quoted here. Other producers such as Oud Beersel report using higher hopping rates <ref> Conversation between Dave Janssen and Gert Christiaens of Oud Beersel, 19-September-2015 </ref>. The use of significantly lower hopping rates may result in less bacterial inhibition and lead to different types of bacteria present. Some lambic producers are experimenting with the use of fresh dried dried hops in addition to or instead of aged hops <ref> [http://www.cantillon.be/br/3_108 Cantillon Iris]</ref> <ref>[https://www.facebook.com/permalink.php?story_fbid=1004839069568069&id=110627652322553 Cantillon Facebook Page post 22-Sept-2015]</ref> <ref>Conversation between Dave Janssen and Jean van Roy of Cantillon, 17-Sept-2015</ref>. James Howat of Black Project Spontaneous Ales uses 0.5 ounces of aged hops per gallon of beer for spontaneously fermented beers brewed using traditional lambic techniques <ref>[https://www.facebook.com/blackprojectbeer/videos/580667305468055/ Howat, James. Facebook live video stream. 12/23/2016. ~5:30 minutes in.]</ref>.

Traditional spontaneous brewers use high hopping rates of aged hops in a long boil. The high For hopping techniques/rates help to regulate bacterial activity and select for the desired bacteria. Aging of the hops lowers the flavor/aroma impact the hops provide and also lowers the bitterness. The aged ops still do provide some bitterness as both oxidized alpha acids and oxidized beta acids can contribute to perceived bitterness and measured IBUs <ref name="OSU talk at CBC 2015"> Understanding How to Control Flavor and Aroma Consistency in Dry Hopped Beer. Dan Vollmertiming, Dan Sharp, Dr. Tom Shellhammer (Oregon State University). Oral presentation at the 2015 Craft Brewers Conference)</ref>. Cantillon uses hops that are on average 2-3 years old at hopping rate of 250-300g/100 L (3.34-4.0 oz/gal)(<ref name="Spontaneous Sour Hour">see [[http://www.thebrewingnetwork.com/membersarchive/sourhour2015_05_wildfriendship.mp3 The Sour Hour Episode 11 with Rob Tod and Jason Perkins from Allagash, Jean Van Roy from Cantillon, and Vinnie Cilurzo from Russian RiverHops#Aged_Hops_in_Lambic|Hops in Lambic]]</ref> ~49 minutes in).

===Cooling=== 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 are sufficiently have a low of -3.9 to 8°C (25---add cutoff temp--46°F) to sufficiently 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 [[Sour WortingWort Souring]], see also [[Spontaneous_Fermentation#Alternative_Applications_of_Spontaneous_Fermentation|sour wortingAlternative applications of ''Spontaneous Fermentation'']]below). In addition this broadWhatever 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, shallow design maximizes ~54 minutes in). Russian River Brewing Company reported that cooling the wort before moving it into the area 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 available and adding it to the mash <ref>[https://www.stitcher.com/show/craft-beer-brewing-magazine-podcast/episode/226-for inoculation -cantillons-jean-van-roy-brewing-comes-naturally-90577613 Jean Van Roy. Interview with ambient microbesCraft 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]]). On In addition it may act as a homebrew scalesafeguard against ''Clostridium botulinum'' (the bacterium responsible for botulism poisoning, where 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 batch sizes cool more quicklypH 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, a wide shallow pan is though if any reported cases of botulism poisoning from beer exist they are not necessary known to achieve appropriate cooling overnight given appropriate nighttime outdoor temperaturesus. Boil kettles Traditional lambic producers have been fermenting unacidified and similarly shaped vessels are sufficient spontaneously inoculated wort for overnight cooling 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>. Hop acids might also partially inhibit ''Clostridium botulinum'' and some other gram-positive pathogens <ref>[https://patents.google.com/patent/US6251461B1/en Antimicrobial activity of hops extract 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 provide influence the activity of ''Pediococcus'' in a rate 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 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 cooling 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 similar 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 provided by coolships 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 production sized batches 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/10688268531455281601687793192762/?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/ Facebook post by James Howat2017.] </ref>. However The dissolved oxygen levels could increase during the aspect ratio of vessels filling of the barrels, although there is no data on this size limit the inoculation that we know of currently. The dissolved oxygen in the wort , however, could be quickly consumed by ambient microbes due aerobic bacteria and yeast (which then generally produce a low pH environment that is hostile to the lower surface area ''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 given volume compared suggestion; however, it is not known how long it would take ''C. botulism'' to traditional coolshipsgrow 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 pH below 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>.  See also:* [[Coolship]]* [https://www.facebook.com/groups/MilkTheFunk/permalink/1986941961334008/ MTF thread on chilling overnight during warmer than normal temperatures.]* [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.]

====Alternative Approaches to Cooling====Vinnie Cilurzo from Russian River Brewing Company historically approached cooling differently for his spontaneously fermented "Solambic" beer. Instead of racking boiling wort into a coolship, Cilurzo runs the wort through a heat exchanger and cools the wort to 68°F, and transfers it into a [[Horny Tank|horny tank]] that is housed within the same barrel room as his other (non-spontaneously fermented) beers. Cilurzo claims that this has reduced the acidity of the final beer <ref>[https://beerandbrewing.com/podcast-episode-86-russian-river Vinnie Cilurzo. Craft and Brewing Magazine podcast; Episode 86. 06/07/2019.]</ref> (~20 mins in). Some lambic brewers have stopped using coolships to cool wort and instead use a plate chiller and then inoculate by moving the cooled wort into a steel tank that has not been CO₂ purged. See [http://www.lambic.info/Brasserie_Mort_Subite#Brewing_Process Brasserie Mort Subite's brewing process] as an example. Logsdon Farmhouse Ales uses a similar technique for their spontaneously fermented ales <ref>Logsdon, Dave. "Sour Power! A Pro Brewer Spontaneous Fermentation Roundtable by Averie Swanson, Dave Logsdon, James Howat, Jeff Mello, and Trevor Rogers". 2018 HomebrewCon presentation.</ref>. ==Fermentation of Spontaneous Beers== Producers of spontaneously fermented beer typically do not oxygenate their wort <ref name="Beer Temple interview with De Garde"></ref> (~27 minutes in). Visual signs of fermentation (CO2 production, krausen, bubbles, etc.) generally takes 4-7 days, although we have seen reports of up to two weeks <ref name="howat_comeandbrewit">[http://comeandbrewit.libsyn.com/2016/page/2/size/25 "Episode 34- Sour Beer 102", Come and Brew It podcast. Interview with James Howat from Black Project Spontaneous Ales. 01/07/2016 (~40 minutes in).]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1183899281638284/ Conversation with Caleb Buck on MTF about spontaneous fermentation. 11/24/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1189818124379733/?comment_id=1194393553922190&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation on MTF with Dustin Carver on how long his spontaneous fermentation took to start. 12/14/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1213062435388635/?comment_id=1215511598477052&comment_tracking=%7B%22tn%22%3A%22R2%22%7D Conversation on MTF with Mark B. Fry on how long spontaneous fermentation took for him. 01/13/2016.]</ref>. Traditional producers conduct fermentation for a [[File:Homebrew spontaneous fermentation.jpeg|200px|thumb|right|Spontaneous fermentation beginning in a carboy]]long time period (1-3+ years) in wooden vessels. The long fermentation process allows the different microbes present to carry out their slow metabolism of the complex carbohydrates present in the beer, developing the flavors and acidity associated with spontaneous beers (see [[Turbid_Mash#Carbohydrate_Composition_and_Utilization_Through_Fermentation|turbid mashing]] for more) <ref name="Van Oevelen et al., 1976">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1975.tb06953.x/abstract Van Oevelen et al., 1976. Synthesis of aroma components during the spontaneous fermentation of lambic and gueuze]</ref> <ref name="Spaepen et al., 1978">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1978.tb03888.x/abstract Spaepen et al., 1978. Fatty acids and esters produced during the spontaneous fermentation of lambic and gueuze]</ref>. Extended aging in the same vessel as fermentation does not present the same sort of autolysis problems that may be found in 'clean' beers aged for long periods of time on the yeast cake. There are some ideas for why this is the case, such as the extended activity of other microbes taking up autolysis products. It is also possible that the influence of autolysis is found, but that they are expressed differently in these sorts of beers and some say that the autolysis character is an important component of the beers <ref>[http://www.thebrewingnetwork.com/the-sour-hour-episode-9/ Rudy Ghequire from Rodenbach on the Sour Hour]</ref> (~28 minutes in). 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>. 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>[https://www.homebrewtalk.com/media/raj-apte-o2-table.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 (between 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]]). American brewers who use coolships for spontaneous fermentation have reported that the success rate for spontaneously fermented beer is around 90-80%. Brewers will often dump undrinkable beers from individual barrels or even beers from barrels that don't meet the expectations of the brewers <ref name="howat_comeandbrewit" />.  ===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 ProgressBelgian lambic producers, ''Enterobacter'' spp., such as ''Enterobacter aerogenes'', ''Enterobacter cloacae'', ''Enterobacter hormaechei'' and ''Enterobacter kobei'', ''Klebsiella aerogenes'', ''Klebsiella oxytoca'', ''Klebsiella varicola'', ''Escherichia coli'', ''Hafnia alvei'', ''Hafnia paralvei'', and ''Citrobacter freundii'', have been found in lambic, with ''E. cloacae'' and ''K. aerogenes'' as the most frequently found ones. Although these enterobacteria contribute little in terms of gravity drop over the first month of fermentation (they mostly consume sucrose in the wort), they do contribute aroma and flavor compounds and precursors during the initial stages of spontaneous fermentation, particularly acetoin, 2,3 butanediol, acetic acid, lactic acid, succinic acid, DMS, acetaldehyde, long-chain fatty acids (these play a role in both flavor impact and providing nutrients for yeast later in the fermentation process), and small amounts of glycerol, ethyl acetate, and higher alcohols which might form esters in the later stages of fermentation. Enterobacteria can also contribute to the production of [https://en.wikipedia.org/wiki/Biogenic_amine biogenic amines] in fermented foods and beverages, including spontaneously fermented beers. Enterobacteria usually disappear after 30-40 days of fermentation due to the increase in ethanol, decrease in pH, and a decrease in food availability <ref name="Martens et al., 1992" /><ref name="Roos_2018">[https://www.ncbi.nlm.nih.gov/pubmed/30246252?dopt=Abstract Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production. Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>, although one study by Curtin et al. reported finding at least small populations of enterobacteria as late as up to 4.5 months <ref name="curtain_asbc_2018">[https://www.asbcnet.org/lab/webinars/webinars/Pages/funkyFermentationsWebinar.aspx Chris Curtin. ASBC webinar: "Funky Fermentations". 12/12/2018. Retrieved 01/03/2019.]</ref>(~25 minutes in), as well as a significant population of ''Komagataeibacter'', a genera normally found in kombucha, after 135 day <ref name="Curtin_2021">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2020.1795607?journalCode=ujbc20 Avi Shayevitz, Keisha Harrison & Chris D. Curtin (2021) Barrel-Induced Variation in the Microbiome and Mycobiome of Aged Sour Ale and Imperial Porter Beer, Journal of the American Society of Brewing Chemists, 79:1, 33-40, DOI: 10.1080/03610470.2020.1795607.]</ref>.  Acetic acid bacteria (AAB) are also present during the first stage of fermentation before alcoholic fermentation begins. These consist of a large diversity of species from ''Acetobacter'' and ''Gluconobacter'', with different species thriving more than others at different points during the long fermentation of lambic and some species found being different in different casks <ref name="De_roos_AAB_2018">[https://journals.asm.org/doi/10.1128/AEM.02846-17 Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role. ASM Journals. Applied and Environmental Microbiology. Vol. 84, No. 7. DOI: https://doi.org/10.1128/AEM.02846-17.]</ref>, including two species that were first described by studies researching lambic (''Acetobacter lambici'' and ''Gluconbacter cerevisiae'' sp. nov. <ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.057315-0 Acetobacter lambici sp. nov., isolated from fermenting lambic beer. Spitaels, Freek and Li, Leilei and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.057315-0.]</ref><ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.059311-0 Gluconobacter cerevisiae sp. nov., isolated from the brewery environment Free. Spitaels, Freek and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.059311-0.]</ref>, as well as ''Acetobacter cerevisiae'' and ''Acetobacter faborum'' <ref name="Roosa_2024">De Roos, J., Vermotea, L., Cnockaertb, M., Vandammeb, P., Weckxa, S., & De Vuysta, L. WOODEN BARRELS HELP TO STEER THE LAMBIC BEER FERMENTATION AND MATURATION PROCESS.</ref>). Acetic acid bacteria are able to grow for the first few weeks because oxygen is available from filling the casks. Once alcoholic fermentation begins, oxygen becomes limited, and the acetic acid bacteria population greatly decreases. Acetic acid bacteria appear again after the alcoholic fermentation phase <ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. For example, Curtin et al. (2018) reported that acetic acid bacteria came and went at various random points within a 0-4.5 month period of fermentation <ref name="curtain_asbc_2018" />(~26 minutes in). De Ross et al. (2018) reported finding small amounts of acetic acid bacteria in lambic during the first few days of fermentation, which then disappeared once alcoholic fermentation began. AAB then reappeared in the casks in greater numbers at week 7 of fermentation, and continued to be isolated in gradually decreasing cell counts for 24 months, the end of which AAB was no longer isolated <ref name="De_roos_AAB_2018" />.  Acidifying the wort to a pH below 4.5 before cooling and exposing to ambient microbes in a coolship can partially eliminate the enterobacteria phase of spontaneous fermentation and thus avoid or limit biogenic amine production, which is a common practice for some lambic breweries <ref name="Spitaels et al., 2015" /><ref name="Roos_2018_2" />. While enterobacteria and oxidative yeasts are not considered to be a part of the core microbes in spontaneous fermentation, it has been shown that ''Saccharomyces cerevisiae'' is metabolically stimulated when co-fermented with some of these species, allowing the ''S. cerevisiae'' to consume more glucose and nitrogen and to more quickly replicate <ref name="Roos_2018" />. De Roos et al (2018) reported significant populations of the enterobacteria species ''Klebseilla variicola'', ''Klebsiella oxytoca'', and the yeast species ''Hanseniaspora uvarum'', ''Saccharomyces cerevisiae'' during the first week or two of lambic fermentation that was pre-acidified (see [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252343/figure/F3/?report=objectonly Figure 3]). Landschoot et al (2015) sampled lambic wort that was pre-acidified to a pH of 4 after being housed in a coolship overnight and during the early weeks of fermentation and found no ''Enterobacteriaceae'' 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==== The second stage of spontaneous fermentation is dominated by ''Saccharomyces'' species (predominantly ''S. cerevisiae'', ''S. bayanus'', ''S. kudriavzevii'', and ''S. pastorianus'', the latter two species often being present towards the end of this phase in lambic due to the colder cellar temperatures during the winter season when lambic is made). ''Hanseniaspora uvarum'' has also been reported in some but not all lambic fermentations playing a major role in starting the second stage of spontaneous fermentation, which is characterized by ethanol production <ref name="Roosa_2024"/><ref name="Bongaerts_2021" />. Most of the attenuation is accomplished during this stage with the depletion of monosaccharides, disaccharides, and trisaccharides consumed in that order (glucose/fructose is consumed first by the ''Saccharomyces'' species, and then maltose/maltotriose are gradually depleted until they are gone by the end of the second stage). ''S. kudriavzevii'' is capable of breaking down maltooligosaccharides (dextrins) through alpha-glucosidase enzyme production, and therefore can out-compete ''S. cerevisiae'' in the later portion of the second stage <ref name="Bongaerts_2021" />.  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" /><ref name="Bongaerts_2021" />. In addition to the bulk of the overall ethanol production, this phase also sees the production of higher alcohols and the synthesis of esters, especially isoamyl acetate, as well as glycerol, caprylic acid, and capric acid <ref name="Van Oevelen et al., 1977" /><ref name="Roos_2018" />. It has been reported by some brewers that this stage might begin as early as 3-14 days and corresponds with a drop in pH below that of regular beer, indicating that the first stage for some spontaneous fermentations might be shorter and faster than reported in the other literature <ref>[http://www.spontanmanc.co.uk/?p=66 Zach Taylor of Chorlton Brewing Co. "The Lab Work Begins". Spontanmanc blog. 08/01/2018. Retrieved 08/29/2018.]</ref>. MTF members (both homebrewers and professionals) have observed yeast fermentation activity typically at 3-7 days <ref>[https://www.facebook.com/events/666424196868756/ Various MTF members. Milk the Funk - Collaboration Brew #3: Spontaneous. 05/01/2017. Retrieved 08/29/2018.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1571139996247542/?comment_id=1571597289535146 Raf Soef, James Howat, Levi Funk. Milk The Funk Facebook thread on how long it takes for yeast to start fermenting in a spontaneous fermentation. 2017.]</ref>. However, these reports are anecdotal based on visual fermentation and microbe analysis was not done in many cases. De Roos et al. (2018) reported that for wort that is pre-acidified to a pH of 4.5, and after an initial drop in pH to 3.8 by enterobacterial and acetic acid bacteria, the pH rose to 4.0 during the secondary fermentation phase, indicating that the yeast consumed some of the organic acids that were produced during the initial enterobacteria phase <ref name="Roos_2018_2" />. ====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 acidification phase is also accompanied by the growth of acetic acid bacteria (AAB), which can be undesirable if this growth is excessive since it leads to greater [[Acetic Acid|acetic acid]] production (in high quantities, acetic acid smells and tastes like vinegar and is very harsh on the palate and throat) as well as acetoin. These microbes include species from the genera of ''Acetobacter'' and ''Gluconobacter''. The species diversity of these genre is lower than during the primary stage due to acid and ethanol selecting for species that are more tolerant to these harsher conditions. For example, De Roos et al. (2018) reported high numbers of ''Acetobacter pasteurianus'', which contains extra genes that code for acid and ethanol tolerance more so than other species of ''Acetobacter'', in lambic from month 3 to month 6, with it disappearing around month 9-13 as ''Pediococcus damnosus'' took its place. ''Acetobacter lambici'' is another species found in lambic during this stage and is well adapted to the lambic environment due to its ability to break down maltooligosaccharides (dextrins) via maltooligosyl trehalose synthase. These microbes are dependent on oxygen in order to metabolize ethanol into acetic acid (with acetaldehyde produced as an intermediary step) and acetoin from lactic acid and are found on the surface of the wort where oxygen is available. The beer/air interface (or surface of the beer that interfaces with the air above it) is also where higher concentrations of acetic acid, ethyl acetate, and acetoin are found due to the AAB being present there rather than deeper within the beer (this is similar to [[Flanders Red Ale]]) <ref name="Roos_2018_2" /><ref name="Roos_2018" /><ref name="Bongaerts_2021" />. With the flavor threshold of acetic acid in beer being 90 ppm <ref>[https://www.aroxa.com/beer/beer-flavour-standard/acetic-acid Aroxa website. "Acetic Acid". Retrieved 11/19/2018.]</ref>, and the levels of acetic acid in Belgian gueuze/lambic being reported in the range of 727-2240 ppm, acetic acid levels in this range is an important flavor compound in spontaneously fermented beers <ref>[http://beachwoodbbq.com/pdf/BBAIBLTBLENDERY.pdf Ryan Fields. "Brewing Beer in America Inspired By the Belgian Lambic Tradition". 2018.]</ref><ref name="Spitaels et al., 2015" />. During a second phase of growth of acetic acid bacteria starting at week 7 in lambic casks, significantly more acetoin (moldy/must flavor when above 50 ppm <ref>[https://www.morebeer.com/articles/Fatty_Flavors_Diacetyl "Fatty Flavors and Diacetyl - Should Your Beer Be Fat-Free?". MoreBeer website. Scott Bickham. Retrieved 04/04/2023.]</ref>) was found in the top portion of the lambic casks above flavor threshold. Acetic acid bacteria has been shown to reduce lactic acid into acetoin, and in another study by De Roose et. al (2023) the researchers found that ''Acetobacter lambici'' present in lambic samples that were tested had the genetic capability to utilize lactic acid as a food source <ref name="Roosa_2024"/>. Acetoin was gradually reduced (presumably metabolized by ''Brettanomyces'') below flavor thresholds at month 9 until it reached near 0 ppm around month 18 <ref name="De_roos_AAB_2018" />. ====Fourth Stage: Maturation==== The fourth and last phase of spontaneous fermentation, also known as the extended maturation phase, is dominated by ''Brettanomyces'' yeast, which is a genus of yeasts that are highly tolerant of low pH, high alcohol, and can survive in low-nutrient conditions (see ''[[Brettanomyces]]'' for more information), as well as lactic acid bacteria from the genera ''[[Pediococcus]]'' and to a lesser extent ''[[Lactobacillus]]'' and certain other yeast species. The most abundant species of ''Brettanomyces'' found in spontaneously fermented beer are strains of ''B. bruxellensis'' (''B. lambicus'' is often found, but has been reclassified as a strain of ''B. bruxellensis''). ''B. bruxellensis'' was first isolated from English stock ales in 1904 and then first isolated from lambic in 1921 by Belgian researchers Kufferath and Van Laer. ''B. anomalus'' and ''B. custersianus'' have also been found, but to a lesser extent than ''B. bruxellensis''. ''Pichia membranifaciens'', ''Debaryomyces hansenii'', and ''Wickerhamomyces anomalus'' are examples of other yeast species that have been found to a lesser extent in lambic during the maturation phase <ref name="Van Oevelen et al., 1977" /><ref name="Roos_2018" /><ref name="Roos_2018_2" /><ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. In some but not all lambic, a shift from ''B. bruxellensis'' to ''B. custersianus'' can occur during the end of maturation, which seems to occur in more porous barrels that have more oxygen ingress and correlates with its preference for a more aerobic environment <ref name="Bongaerts_2021" />.  This phase generally begins somewhere around month four to eight, with these microbes completely dominating at around 9-13 months <ref name="Roos_2018_2" /><ref name="curtain_asbc_2018" />(~26 minutes in). Additional attenuation occurs very slowly for another 7-18 months. De Roos et al. (2018) reported a gradual drop from 4 Plato to 0.5 °Plato during the maturation phase. <ref name="Roos_2018_2" />. During this extended maturation phase, ''Brettanomyces'' continues to ferment the residual sugars leftover in the beer using intra- and extracellular alpha-glucosidase, and produces most of the final aromatic and flavor compounds in the form of esters, phenols, and fatty acids found in finished Belgian lambic and other spontaneously fermented beers (see [[Brettanomyces#Brettanomyces_Metabolism|''Brettanomyces'' metabolism]]). During the maturation phase, a [[pellicle]] is formed from the ''Brettanomyces'', as well as oxidative yeasts from the genera ''Pichia'', ''Candida'', ''Cryptococcus'', and ''Torulspsis'' <ref name="Van Oevelen et al., 1977" /><ref name="Roos_2018" /><ref name="Roos_2018_2" /><ref name="Bongaerts_2021" />. It is thought that the pellicle and the presence of these oxidative yeasts might reduce oxygen influx, and thus assist in inhibiting the growth of acetic acid bacteria <ref>[https://pdfs.semanticscholar.org/8c12/9985b9f1264179fe2e2f779bae1ff3e51a54.pdf Jacques De Keersmaecker. "The Mystery of Lambic Beer". Scientific American, Inc. 1996.]</ref>, however, this has not been proven in a scientific manner that we know of. During 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://www.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 ''Pediococcus'' strains are known to produce. These exopolysaccharides can be broken down by other microbes present in the beer relieving the beer of its "sickness" (this exopolysaccharide breakdown is generally attributed to ''Brettanomyces''). Beer may also become "sick" in the bottle during bottle conditioning. This is likely due to enhanced ''Pediococcus'' activity from additional fermentable sugar, in the form of simple sugars or beer which has not completely attenuated yet <ref name="Vinnie sour beer talk">[http://www.thebrewingnetwork.com/post1863/ Recording of Vinnie's talk at NHC]</ref> (~1:47 in). A beer that 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.  Acetic acid bacteria tend to be absent from bottled gueuze due to the anaerobic environment. ''Brettanomyces'' and ''Pediococcus damnosus'' are often recoverable from bottles of gueuze as old as five years, but ''Pichia membranifaciens'' and ''Saccharomyces cerevisiae'' have also been recovered from two year old bottles of gueuze. After five years of storage, lactic acid bacteria was no longer found in bottled gueuze <ref name="Bongaerts_2021" />.  See also:* [http://www.lambic.info/Microbiology_and_Biochemistry Lambic.info "Microbiology and Biochemistry" wiki page].* [http://beachwoodbbq.com/pdf/BBAIBLTBLENDERY.pdf "Brewing Beer in America Inspired By the Belgian Lambic Tradition" by Ryan Fields, former head brewer and blender at Beachwood Blendery.]* [https://www.facebook.com/groups/MilkTheFunk/permalink/4224750307553151/ MTF post by Zach Taggart gives an example of plating out microbes for different time points during spontaneous fermentation at 42 North Brewing Company.] ===Aroma and Flavor Production===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>.  Below is a collection of data on spontaneously fermented beer sampled from bottles available in the market; see the data 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) || 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. (2017) <ref name="witrick_2017" />|-| Cantillon Gueuze (vintage not reported) || 3.44 || 3.29 || 1417 || 1224|| Witrick et al. (2017) <ref name="witrick_2017" />|-| Cantillon Bio (vintage not reported) || 3.53 || 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, Bottle 2 || - || - || 3800 || 1050 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2013, Bottle 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" />|-| Cantillon Gueuze; Bottled 2010, Bottle 2 || - || - || 8500 || 2020 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, Bottle 3 || - || - || 10600 || 2240 || Spitaels et al. (2015) <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, Bottle 2 || - || - || 13800 || 1270 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, Bottle 3 || - || - || 11900 || 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, Bottle 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 || - || 8.6 || - || 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, Bottle 3 || - || - || - || - || - || 11.3 || - || 195 || 130 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2011, Bottle 1 || - || - || - || - || - || 3.3 || - || 166 || 170 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| 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. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, Bottle 1 || - || - || - || - || - || 2.9 || - || 249 || 203 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, Bottle 2 || - || - || - || - || - || 8.2 || - || 295 || 283 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2010, Bottle 3 || - || - || - || - || - || 6.5 || - || 332 || 285 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2008, Bottle 1 || - || - || - || - || - || 2.2 || - || 371 || 125 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| 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. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, Bottle 1 || - || - || - || - || - || 2.0 || - || 630 || 140 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, Bottle 2 || - || - || - || - || - || 3.4 || - || 592 || 197 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 2004, Bottle 3 || - || - || - || - || - || 2.7 || - || 464 || 178 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, Bottle 1 || - || - || - || - || - || 1.8 || - || 526 || 89 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, Bottle 2 || - || - || - || - || - || 3.9 || - || 778 || 150 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-| Cantillon Gueuze; Bottled 1996, Bottle 3 || - || - || - || - || - || 2.5 || - || 556 || 169 || Spitaels et al. (2015) <ref name="Spitaels_2015_Bottles" />|-|} ===Biogenic Amines===[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 example, 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>[https://www.mdpi.com/2304-8158/8/2/62/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>. Some countries have set an upper limit of histamine in wine to be anywhere from 2-10 mg/l <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.]</ref>. Loret et al. (2005) quotes the upper limit of individual amines to generally be safe for consumption at below 10 mg/l <ref name="loret_2005" />.  Loret et al (2005) examined the levels of biogenic amines in Belgian beers, including lagers, traditional ales, bottle conditioned ales, and spontaneously fermented beer (presumably lambic). They found that spontaneously fermented beers, which were 42 samples out of the total 297 samples and from 10 different breweries, generally contained the most biogenic amines, namely tyramine (associated with hypertension), histamine (associated with hypotension), and cadaverine. However, not all of these breweries produced higher levels of amines, suggesting that processing in some breweries is limiting the biogenic amine production (this process was not identified in the Loret et al study, but the process is likely to be the lowering of the wort pH to 4.5 to limit the enterobacteria phase). For example, 6 of the spontaneous fermentation breweries had levels of histamine as an average between 20-45 mg/l, and levels of tyamine between 30-60 mg/l, which is higher than the upper limit that is generally considered to be safe for consumers (this upper limit was stated as 10 mg/l by Loret et al.). The other 4 breweries had levels of histamine and tyramine between 0-20 mg/L, as an average. Interestingly, they also found that a small number of ales and bottle conditioned ales, 21 out of 220 samples, also had levels of tyamine above the upper limit of 10 mg/l. None of the lagers sampled had biogenic amines above 10 mg/l <ref name="loret_2005" />. Another study in Portugal measured biogenic amines in 5 Portuguese craft beers (4 ales and 1 lager; no mixed fermentation or sour beers) and reported that the total biogenic amine content was less than 10 mg/l for all of the beers <ref>[https://www.mdpi.com/1424-8220/23/1/343 Gil, R.L.; Amorim, C.M.P.G.; Amorim, H.G.; Montenegro, M.d.C.B.S.M.; Araújo, A.N. Influence of Brewing Process on the Profile of Biogenic Amines in Craft Beers. Sensors 2023, 23, 343. https://doi.org/10.3390/s23010343.]</ref>.  De Roos et al. (2018) measured biogenic amines over the fermentation lifespan of Belgian lambic beers that were pre-acidified to a pH of 4.5 before being cooled in a coolship and spontaneously fermented. They found that the initial wort had low concentrations of some biogenic amines, such as agmatine (9 mg/l), putrescine (8 mg/l), and cadaverine (3 mg/l). In one cask, the agmatine remained stable while in the second cask the agmatine declined to zero during the maturation phase, and then slightly increased to less than 5 mg/l. Cadaverine was produced during the first three weeks of fermentation and remained steady throughout the fermentation process at about 30 mg/l. Histamine was produced during the acidification phase by ''Pediococcus damnosus'' between 3 and 9 months and ended up at around 15 mg/l. Tyramine had final concentrations of around 30-40 mg/l and was formed either during the acidification phase (6 months) or the late maturation phase (18-24 months), potentially by ''P. damnosus'' or some other LAB that was at too low of a population to detect, or maybe as a result of autolysis of dead yeast cells. 2-Phenylethylamine and tryptamine were never found in the lambic beers <ref name="Roos_2018_2" />. A second study by the same group of researchers in 2024 confirmed that ''P. damnosus'' was responsible for producing histamine in lambic samples that were tested at one brewery; the total biogenic amines remained below the regulatory levels deemed safe for consumption in Europe <ref name="Roosa_2024"/>. The biogenic amines content of spontaneously fermented beer that is not acidified before fermentation is not likely to be high enough to cause serious physical harm. Large amounts of beer must be consumed before advisable limits are reached. However, the additive effect of different amines consumed with a large amount of beer might cause health related reactions in some people. See [http://suigenerisbrewing.com/index.php/2019/01/22/biogenic-amines/ "Fact or Fiction – Biogenic Amines in Beer" by Dr. Bryan Heit; an analysis of biogenic amines in spontaneously fermented beer and associated health concerns] for more information. For more information on biogenic amines, see the following:* [http://www.who.int/iris/bitstream/10665/89216/http://apps.who.int//iris/bitstream/10665/89216/1/9789240691919_eng.pdf "Public Health Risks of Histamine and other Biogenic Amines from Fish and Fishery Products," FAQ on biogenic amines by the World Health Organization.]* [[Brettanomyces#Biogenic_Amines|Biogenic amines produced by ''Brettanomyces'']].* [http://www.horscategoriebrewing.com/2019/01/spontaneous-fermentation-and-biogenic.html "Spontaneous fermentation and biogenic amines" by Dr. Dave Janssen; review of several studies that looked at the levels of biogenic amines in different beers and their production, and their potential flavor contribution.]* [http://suigenerisbrewing.com/index.php/2019/01/22/biogenic-amines/ "Fact or Fiction – Biogenic Amines in Beer" by Dr. Bryan Heit; an analysis of biogenic amines in spontaneously fermented beer and associated health concerns.]** [https://www.facebook.com/groups/MilkTheFunk/permalink/2570617069633158/?comment_id=2570641549630710&reply_comment_id=2570664762961722&comment_tracking=%7B%22tn%22%3A%22R8%22%7D Follow up from Dr. Heit on MTF on "allergic-like" reactions to biogenic amines.]* [http://suigenerisbrewing.com/index.php/2020/07/07/nitrosamines-fact-or-fiction "Fact or Fiction Part VI – Nitrosamines in Beer," by Dr. Bryan Heit.]* [https://www.youtube.com/watch?v=GZoHGWuMeEk "The surprising truth about histamine intolerance - Dr. Will Bulsiewicz," ZOE Podcast.] ===Source of Microbes===The sources of microbes that influence spontaneous fermentation are somewhat debatable, especially when referencing [[lambic]] producers in Belgium. Some Belgian lambic brewers have made claims historically that microbe populations in the valley of the Senne River are unique enough that spontaneously fermented beer cannot be made anywhere else; however, American Coolship Ales and scientific inquiry have demonstrated that the genus level microbes that ferment spontaneously fermented beer are very similar in other parts of the world (although species and strain differences might have an impact on specific flavor profile differences between different regions of the world) <ref name="curtain_asbc_2018" /><ref>[https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035507 Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale. Bokulich NA, Bamforth CW, Mills DA (2012) Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale. PLOS ONE 7(4): e35507. https://doi.org/10.1371/journal.pone.0035507]</ref>. Although the coolship step is regarded by lambic brewers as the main contributor to the microbial inoculation for spontaneous beers, it has been demonstrated that ''Brettanomyces'' and other microbes living in the barrels are at least partially responsible for the secondary fermentation of spontaneous beers. Some yeast species that have been found in limited capacity in lambic such as ''Wickerhamomyces anomalus'' and ''Debaryomyces hansenii'' have been isolated from the air of coolship rooms, but they have also been isolated from the air of cellar rooms, indicating that they could have been introduced to the wort/beer in the cellar as well. Additionally, yeast and bacteria species that are the most abundant during lambic fermentation (''Saccharomyces'', ''Brettanomyces'', ''Pediococcus'', and acetic acid bacteria) have not been isolated from the air of coolship rooms <ref name="Bongaerts_2021" />. De Roos et al. (2018) used amplicon sequencing technology (which is better at detected so-called "[[Quality_Assurance#Viable_But_Nonculturable|viable but not culturable]]" cells) to sample of the interior of barrels and foeders used in one lambic brewery that uses high-pressurized hot water and sulfur dioxide to clean them, and found surviving colonies of ''Brettanomyces anomalus'', ''B. bruxellensis'', ''B. custersianus'', ''Pediococcus'', and to a lesser extent ''Acetobacter'' and ''Lactobacillus''. The ''Acetobacter'' might be linked to older barrels that are more porous. ''Pichia'', ''Debaryomyces hansenii'' and ''Candida'' were also found in abundance in some barrels, but not others. They also found high levels of ''Cellulosimicrobium'' and ''Acinetobacter'', which have not been found to be important for lambic fermentation; the authors suspected that these were living in the wood rather than in the beer since they can metabolize cellulose. Although they are probably also not important to the fermentation of lambic, molds were found in the more porous barrels before cleaning, including ''Aspergillus'' and ''Penicillium''. No molds survived the sulfuring process and the diversity of microbes was far higher in barrels than it was for foeders, perhaps because of the higher level of oxygen exposure in barrels compared to foeders, although foeders had a much higher than expected amount of surviving ''Saccharomyces'' and ''Pichia'' <ref>[https://aem.asm.org/content/early/2018/10/15/AEM.02226-18 The interior surfaces of wooden barrels are an additional microbial inoculation source for lambic beer production. J. De Roos, D. Van der Veken, L. De Vuyst. 2018. DOI: 10.1128/AEM.02226-18.]</ref>.  Professor Chris Curtin from Oregon State University presented the results of a study by his team where 50 barrels of three different vintages of spontaneously fermented, lambic-inspired beer was sampled three times over a 9 month period. These 50 barrels represented three batches of the same recipe brewed in 2013, 2015, and 2016. With the exception of a small number of barrels, they all contained the same or similar ''Brettanomyces bruxellensis'' yeast. There was no significance between the three different batches. Two of the 2015 barrels had a different species of ''Brettanomyces'' (''claussenii''). Two of the 2016 barrels had more ''Saccharomyces cerevisiae''. This study indicated that barrel to barrel variation as far as yeast goes is fairly small at this brewery. Bacteria populations clustered much closer based on the vintage (but not the barrel). The 2013 batch was dominated by ''Gluconobacter'' and ''Acetobacter'', while the 2015 batch was dominated only by ''Acetobacter'', and the 2016 batch was dominated by ''Lactobacillus''. This indicated that the vintage of the batch plays a major role in determining which bacteria will be the dominant bacteria during maturation, but individual barrels generally do not (although a small number of barrels matured faster than the majority of the barrels and were dominated by bacteria that represented the more mature vintage). Despite the lack of major variation between barrels, Curtin determined that some barrels can introduce microbial variation, perhaps due to insects transferring microbes or differences in the oxygen ingress between different barrels, and the ability of yeast and bacteria to live within the inner surface of the barrels and potentially survive cleaning procedures <ref name="curtain_asbc_2018" /> (~29 minutes in)<ref name="Curtin_2021" />.  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 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 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. Other sources for the microbes include the ceilings of the breweries which are often not cleaned in Belgian lambic breweries, and the coolship itself. Some breweries have opted to craft wooden overhangs to simulate having a relatively low ceiling above the coolship (see [[Coolship#Overhangs_and_Other_Innovations|Coolship]]). As the wort cools overnight, vapor rises and condenses on the wooden ceiling or overhang above, and then drips back down into the coolship carrying any microbes that might be living on the surface of the wood. The air also carries some amount of microbes, although it is rare to find wild ''Brettanomyces'' in the air (see [[Brettanomyces#Environment_and_Survival|''Brettanomyces'' environment]]). The sources for various microbes are unknown and potentially many, and probably vary from producer to producer. Additionally, no single source is likely to be required to have a successful spontaneous fermentation. 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'' 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 being used with water, potassium metabisulfite, and citric acid). Andy reported that the beer was also not sour and did not have a ''Brettanomyces'' 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. ==Alternative Applications of Spontaneous Fermentation== Much of the above discussion has focused on spontaneous fermentation as applied to lambic and lambic-inspired brewing. Some brewers are applying spontaneous fermentation to yield beers quite different from lambic-oriented brewers. A notable example of this is [[De Garde]], whose entire lineup of beers are cooled in a coolship and don't see pitched yeast <ref name="Beer Temple interview with De Garde">[https://vimeo.com/127084279 The Beer Temple Interviews #264 with Trevor Rogers of De Garde]</ref> (excepting perhaps a bit of pitched yeast in some beers for bottling conditioning). De Garde produces a range of spontaneous beers including beers similar to Berliner weisse by warm incubation after spontaneous inoculation <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/814831538545062/ MTF facebook conversation with a screenshot of brief De Garde process, March 2014]</ref>. By manipulation of parameters such as grist, hopping levels and incubation/fermentation temperatures, a diverse range of beers of spontaneous fermentation can be produced outside of lambic-inspired beers. Jester King has put grapefruit zest in the coolship and run hot wort through ~1 lb/bbl of Sorachi Ace before the coolship<ref name='Jester King on the Sour Hour pt. 1'>[http://www.thebrewingnetwork.com/the-sour-hour-episode-14/ The Sour Hour #14: with Jester King, pt. 1]</ref> (~57 minutes in). ===Re-pitching the Microbes from Spontaneous Fermentation=== Brewers can also harvest the slurry of a spontaneous fermentation or a portion of the beer itself to inoculate future batches of beer. Although there are potentially hundreds of microbes initially in a spontaneous fermentation, after a few months there will only be a few groups which are able to survive the harsh conditions of sour beer. These include potentially ''Saccharomyces'', ''Brettanomyces'', lactic acid bacteria, and perhaps ''Pichia''. Therefore, an aged slurry from spontaneous fermentation can be treated the same as re-using a [[Mixed_Fermentation#Reusing_a_Sour_Yeast_Cake|mixed fermentation]] yeast cake. A [[Solera]] approach should also be a successful approach. It isn't clear on if using a slurry from a spontaneous fermentation to inoculate a new batch of wort/beer would constitute the new batch being called a "spontaneously fermented beer". From a process perspective, given the adopted definition of "spontaneous fermentation" as described in this wiki article, re-using slurries from a spontaneous fermentation is fundamentally a different process. Since it is a fundamentally different process, it appears to be logical that the resulting new beer would not be a "spontaneously fermented" beer. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1082179065143640/ this discussion with James Howat on MTF] as an example of how brewers are approaching this terminology. ===Supplimenting a Spontaneous Fermentation=== Another related process is to supplement a spontaneous fermentation which is carried out as defined by this wiki article but to also pitch a commercial lab pitch of microbes, cultured wild caught microbes or a slurry from a previous spontaneous fermentation. These options might be necessary for failed attempts for a spontaneous fermentation to successfully start fermenting with the desired week or two. Pitching lab cultures might result in a different flavor result than if the wort was fermented completely spontaneously since lab cultures will often out-compete and eventually kill many other species of microbes that would be present in wort that was cooled overnight in a coolship. As with pitching microbes from a previous spontaneous fermentation, these processes are a deviation from the 100% spontaneous fermentation process, and therefore are sometimes not referred to as "spontaneous fermentation", but perhaps "partial spontaneous fermentation" or some other term. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1212505245444354/?comment_id=1212528145442064&comment_tracking=%7B%22tn%22%3A%22R%22%7D this MTF debate] on the effects of supplimenting spontaneous fermentation with cultured microbes. ===Spontaneous Fermentation From Fruit===Traditional wine and cider making techniques include not adding any lab microbes, but only relying on the microbes found on the surface of the fruit. This could be considered as a subtype of spontaneous fermentation that is distinct from using a coolship to inoculate wort but is still reliant on the native microflora of the fruit. Rather than a subtype of spontaneous fermentation, this method has also been considered to be more appropriately referred to as "wild inoculation" or "non-selected yeast fermentation" <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2125624937465709/?comment_id=2125655434129326&reply_comment_id=2126302030731333&comment_tracking=%7B%22tn%22%3A%22R%22%7D Justin Amaral. Milk The Funk Facebook group discussion on inoculating wort with fruit. 06/12/2018.]</ref><ref name="ahmed_fruit">[https://www.facebook.com/groups/MilkTheFunk/permalink/2125624937465709/?comment_id=2126549127373290&reply_comment_id=2129101360451400&comment_tracking=%7B%22tn%22%3A%22R%22%7D Tariq Ahmed. Milk The Funk Facebook group discussion on inoculating wort with fruit. 06/14/2108.]</ref>. It is believed by some cider makers that the source for native microbes in their fermentations often comes from the equipment being used to process the cider apples rather than the apples themselves, although there hasn't been enough research in this area to know for sure if the major source for native microbes comes from the apples or the equipment used in cider making <ref name="ahmed_fruit" />.  See also:* [[Cider#Spontaneous_Fermentation|Cider]]* [[Wine]]* [https://www.facebook.com/groups/MilkTheFunk/permalink/2125624937465709/ MTF thread on using large amounts of wine grapes or other fruit to inoculate wort.] ==Spontaneous Fermentation versus Mixed Fermentation== Spontaneous fermentation yields the greatest diversity of microbes in the wort, including many outside of ''[[Saccharomyces]]'', ''[[Brettanomyces]]'', ''[[Pediococcus]]'' and ''[[Lactobacillus]]'' <ref name="Bokulic et al., 2012" /> <ref name="Spitaels et al., 2015" /><ref name="Spitaels et al., 2014" />. The degree to which these diverse microbes present during spontaneous fermentation are active and influence the characteristics of the final product is unknown, but brewers report in some cases upwards of 100 distinct microbes present and 24 different microbes which are active and important in producing the character of their beers <ref name="Spontaneous Sour Hour" />(~36 minutes in). Spontaneous fermentation may be conducted anywhere, though the microbes present in different environments and/or at different times of the year or from different cooling rates due to different ambient night time temperatures may be better or more poorly suited for producing a good tasting final product <ref name="Beer Temple interview with De Garde">[https://vimeo.com/127084279 The Beer Temple Interviews #264 with Trevor Rogers of De Garde]</ref> <ref name="Spontaneous Sour Hour" />(~39 minutes in, ~54 minutes in). In addition, many of the microbes active in commercial spontaneous fermentation derive from the brewery environment <ref name="Bokulic et al., 2012" />, which is a benefit that the average homebrewer likely does not have. This great range in the potential of spontaneous fermentation can produce some of the most complex beers in the world, but it can also produce undrinkable products.

Scientific research Some brewers may opt for the more controlled techniques of [[Mixed Fermentation]] to approach the sorts of characteristics found in Belgium spontaneously fermented beers. Mixed fermentation employs the controlled pitching of different lab sourced microbes or bottle dregs. These may be pitched all at once or staggered to control the final product. This greater degree of control can limit some of the risk of poor outcomes and the US has shown can allow a regular general pattern brewer to better achieve the beer they want; however this approach cannot yield the same microbial succession diversity of spontaneous fermentation spontaneously fermented beer. <ref name="Van Oevelen et al.For this reason, 1977">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1977.tb03825.x/abstract/ 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/ Spitaels et al., 2014]</ref> <ref name="Spitaels et al., 2015" />. This as been illustrated well by Raj Apte <ref>[http://www2.parc.com/emdl/members/apte/slides_nchf.pdf/ Raj Apte Concepts homebrewers may need to decide what degree of risk they are willing to take and what sort of sour Beer, 2004]</ref>. The first stage, final product they are after to determine which lasts technique is right for approximately 1 month <ref name="Van Oevelen et althem.Many use a hybridized approach of the two, 1977" /> <ref name="Martens et al., 1992">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1992.tb01126combining open cooling for spontaneous inoculation with pitching of labs cultures and bottle dregs.x/abstract/ Martens et al., 1992]</ref>, While this is dominated by ''enterobacteria''. Though ''enterobacteria'' contribute little in terms of gravity drop over the first month of technically not spontaneous fermentationand it may yield different results from truly spontaneously fermented beers, they may contribute aroma and flavor compounds and precursors during it can be a good balance of the initial stages benefits of spontaneous fermentation <ref name="Martens et al., 1992" />. Acidifying the wort (collection of ambient microbes to pH = 4 before cooling express regional terroir and exposing to ambient a greater diversity of microbes present) with benefits of mixed fermentation (some pre-screening and greater control in dominant microbes to help select for a coolship can eliminate final beer of the brewer''enterobacteria'' phase s preference). Ultimately the brewer must decide which approach, or combination of spontaneous fermentation <ref name="Spitaels et al.the two approaches, is right for them with regard to the desired flavor and aroma profile, adherence to tradition, timeframe, 2015" />and risk of bad beer.

The second stage of spontaneous fermentation is dominated by ''Saccharomyces sp.'' (predominantly ''S. cerevisiae'' and ''S. bayanus''). Most of the attenuation is accomplished during this stage, which lasts approximately 3-4 months ==Media=====Videos===<youtube height="200" width="300">ncWape6UKKQ<ref name/youtube><youtube height="200" width="Van Oevelen et al., 1977300" >MWLvqOv7Vf0</youtube>.

* [https://www.facebook.com/TheBruery/videos/3462243510455831/ The ''Saccharomyces'' dominated stage of Bruery hosted Facebook live panel on spontaneous fermentation is followed by prolonged and gradual acid and flavor development accompanied by the final points of attenuation. In some descriptions this is split into an "acidification phase" which is dominated by lactic acid bacteria (LAB)with Jason Perkins from Allagash, primarily ''Pediococcus''Jim Crooks from Firestone Walker, and a "maturation phase" driven by ''Brettanomyces'' <ref name="Van Oevelen et al.Jeff Stuffings from Jester King Brewery, 1977" />. Other sources describe these as one extended maturation phase with acidification Bob Kunz from ''Pediococcus'' and ''Brettanomyces'' growth occurring simultaneously <ref name="Spitaels et al.Highland Park Brewery, 2015" /> <ref name="Bokulic et alBrandon Jones from Yazoo Brewing Co., 2012" /> <ref name="Spitaels et al., 2014" />. Note that many scientific publications use the terminology ''Dekkera'' rather than ''Brettanomyces''and Keith Pumilia from Brewery Terreux. ----continue this]

During ===Podcasts===* [http://www.thebrewingnetwork.com/the extended maturation phase, a beer may become "sick" or "ropey", though not all producers get this <ref name="Spontaneous -sour-hour-episode-11/ The Sour Hour" /> maybe ~1:10 min in? (also Vinnie on the sunday session podcast detailing spontaneous fermentation process with Rob and maybe lambic summit). This is the result of exopolysaccaridesJason from Allagash, Jean Van Roy from Cantillon, which some ''Pediococcus'' strains are known to produceand Vinnie from Russian River. These exopolysaccharides can be broken down by other microbes present in the beer relieving the beer of its "sickness" (this exopolysaccaride breakdown is generally attributed to ''Brettanomyces'')]* [https://beerandbrewing. A beer may also become "sick" in the bottle during bottle conditioning. This is likely due to enhanced ''Pediococcus'' activity from additional fermentable sugar, in the form com/podcast-episode-103-harrison-mccabe-of simple sugars or beer which has not completely attenuated yet. -beachwood-sources for thisblendery-creating-. A beer which is sick in the bottle will generally clear through / Craft Beer and Brewing Podcast: Harrison McCabe of Beachwood Blendery; Creating the same process as a younger aging beer wen given appropriate timeRight Environment for Spontaneous and Pitched Culture Wild and Sour Beers.]

==''Spontaneous Fermentation'' versus ''Mixed Fermentation''History==(To do) - talk about the differences between '* [http://www.garshol.priv.no/blog/390.html Lars Garshol'spontaneous fermentation'' and [[Mixed Fermentation]], and s write up on the arguments for using different methodshistory of people reusing yeast as opposed to spontaneously fermenting.]

* [http://www.archaicpursuit.com/2018/08/2017-coolship-experiment-hopping-rate.html?m=1 Caleb Buck's collected data on cooling rates, acidity from hopping rates, and other collected data over a multi-year, multi-batch experiment.]* [http://www.stitcher.com/podcast/the-brewing-network/the-sour-hour/e/the-sour-hour-episode-11-37950117 The Sour Hour, Episode 11 with Rob and Jason from Allagash, Jean Van Roy from Cantillon, and Vinnie from Russian River.].* [http://thebrewingnetwork.com/shows/751 Jean Van Roy on The Brewing Network's Sunday Session.]* [http://hwcdn.libsyn.com/p/e/a/2/ea26e00136fe1638/bbr05-30-13cantillon.mp3?c_id=5723890&expiration=1432340356&hwt=a3b044a37355912b46e56da5c64929e5 Jean Van Roy from Cantillon on Basic Brewing Radio.]* [https://www.youtube.com/watch?v=l4i1fyYqIlM&list=PL662949708E13A207 The Lambic Summit: Discussions of lambic with Jean Van Roy of Cantillon, Armand Debelder of 3 Fonteinen, and Frank Boon of Boon.]* [http://www.lambic.info/An_Overview_of_Lambic#Brewing_Lambic Lambic.info page on lambic production.]* [http://www.themadfermentationist.com/2011/04/ambient-spontaneous-yeast-starters.html The Mad Fermentationist on spontaneous yeast starters.]* [http://suigenerisbrewing.blogspot.ca/2013/04/anatomy-of-wild-ferment.html Anatomy of a Wild Ferment; Sui Generis Blog.]* [http://horscategoriebrewing.blogspot.com/2016/02/thoughts-on-spitaels-and-van.html "Thoughts on Spitaels and Van Kerrebroeck et al, 2015", by Dave Janssen on Hors Catégorie Blog - examines microbial and flavor compound evolution over time in bottles of Belgian lambic from Cantillon.]* [https://www.facebook.com/groups/MilkTheFunk/permalink/1300046996690178/ Brandon Jones's notes from CBC 2016 talk on spontaneous fermentation.]* [http://sourbeerblog.com/jester-king-spon-2016-methode-gueuze-tasting-and-interview/ "Jester King 2016 SPON — Méthode Gueuze – Tasting and Interview" on Sour Beer Blog (includes details on process and blending).]* [http://suigenerisbrewing.com/index.php/2018/12/17/coolship-homebrew/ "Going Wild – Coolshipped Beers in the Home Brewery" by Bryan Heit; introduction to spontaneous fermentation with a simpler brewing process (not turbid mashing).]* [https://www.facebook.com/groups/MilkTheFunk/posts/6666386556722835/ List of US breweries that do spontaneous fermentation.]