Changes

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

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" />.

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" />.

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" />.

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" />.

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" />.

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>