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Although spontaneous ales have a common pattern of fermentation by groups of genera of microbes, the diversity in specific species is large across different lambic producers and American spontaneous ale producers (although data for American spontaneous ale producers is limited). In American spontaneous ale producers, ''Klebsiella'' spp., ''Enterobacter'' spp.,'' Pectobacterium carotovorum'', and ''Serratia ureilytica'' have been found. In Belgian lambic producers, ''Enterobacter'' spp., such as ''Enterobacter aerogenes'', ''Enterobacter cloacae'', ''Enterobacter hormaechei'' and ''Enterobacter kobei'', ''Klebsiella aerogenes'', ''Klebsiella oxytoca'', ''Klebsiella varicola'', ''Escherichia coli'', ''Hafnia alvei'', ''Hafnia paralvei'', and ''Citrobacter freundii'', have been found in lambic, with ''E. cloacae'' and ''K. aerogenes'' as the most frequently found ones. Although these enterobacteria contribute little in terms of gravity drop over the first month of fermentation (they mostly consume sucrose in the wort), they do contribute aroma and flavor compounds and precursors during the initial stages of spontaneous fermentation, particularly acetoin, 2,3 butanediol, acetic acid, lactic acid, succinic acid, DMS, acetaldehyde, long-chain fatty acids (these play a role in both flavor impact and providing nutrients for yeast later in the fermentation process), and small amounts of glycerol, ethyl acetate, and higher alcohols which might form esters in the later stages of fermentation. Enterobacteria can also contribute to the production of [https://en.wikipedia.org/wiki/Biogenic_amine biogenic amines] in fermented foods and beverages, including spontaneously fermented beers. Enterobacteria usually disappear after 30-40 days of fermentation due to the increase in ethanol, decrease in pH, and a decrease in food availability <ref name="Martens et al., 1992" /><ref name="Roos_2018">[https://www.ncbi.nlm.nih.gov/pubmed/30246252?dopt=Abstract Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production. Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>, although one study by Curtin et al. reported finding at least small populations of enterobacteria as late as up to 4.5 months <ref name="curtain_asbc_2018">[https://www.asbcnet.org/lab/webinars/webinars/Pages/funkyFermentationsWebinar.aspx Chris Curtin. ASBC webinar: "Funky Fermentations". 12/12/2018. Retrieved 01/03/2019.]</ref>(~25 minutes in), as well as a significant population of ''Komagataeibacter'', a genera normally found in kombucha, after 135 day <ref name="Curtin_2021">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2020.1795607?journalCode=ujbc20 Avi Shayevitz, Keisha Harrison & Chris D. Curtin (2021) Barrel-Induced Variation in the Microbiome and Mycobiome of Aged Sour Ale and Imperial Porter Beer, Journal of the American Society of Brewing Chemists, 79:1, 33-40, DOI: 10.1080/03610470.2020.1795607.]</ref>.
Acetic acid bacteria (AAB) are also present during the first stage of fermentation before alcoholic fermentation begins. These consist of a large diversity of species from ''Acetobacter'' and ''Gluconobacter'', with different species thriving more than others at different points during the long fermentation of lambic and some species found being different in different casks <ref name="De_roos_AAB_2018">[https://journals.asm.org/doi/10.1128/AEM.02846-17 Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role. ASM Journals. Applied and Environmental Microbiology. Vol. 84, No. 7. DOI: https://doi.org/10.1128/AEM.02846-17.]</ref>, including two species that were first described by studies researching lambic (''Acetobacter lambici'' and ''Gluconbacter cerevisiae'' sp. nov. <ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.057315-0 Acetobacter lambici sp. nov., isolated from fermenting lambic beer. Spitaels, Freek and Li, Leilei and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.057315-0.]</ref><ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.059311-0 Gluconobacter cerevisiae sp. nov., isolated from the brewery environment Free. Spitaels, Freek and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.059311-0.]</ref>, as well as ''Acetobacter cerevisiae'' and ''Acetobacter faborum'' <ref name="Roosa_2024">De RoosaRoos, 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>.
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.
A study of spontaneously fermented ales at a brewery in Oamaru, New Zealand, found a greater diversity of dominate organisms at the point of beer maturation. The diversity of microbe species deviated significantly from Belgian lambic beers in general, as well as the diversity between vintages sampled from barrels and between barrels for the same vintage (vintages 2016-2022 CE). The 2016 vintage was completely dominated by ''Pediococcus'' (97.6%); other genera detected included ''Lactobacillus'' (1.24%), ''Komagataeibacter'' (0.18) and Bacillus (0.13%). However, in the 2017 vintage, ''Pediococcus'' was not as predominant, as seen in the 2016 vintage. Here, ''Pediococcus'' made up 33.9%. ''Komagataeibacter'' (22.7%), ''Acetobacter'' (21.7%), and ''Paenibacillus'' (16.3%) were the other common genera associated with this vintage. The 2019 vintage was similar to the 2016 vintage where ''Pediococcus'' showed high relative abundance at 97.5%; however, ''Rhodococcus'' (0.08%) and ''Acetobacter'' (0.33%), which were not detected in previous vintages, were present here in low relative abundance. The 2021 and 2022 vintages were again dominated by ''Pediococcus'', ''Klebsiella'' and members of the Enterobacteriaceae family. The study reported some deviation between barrels sampled for the same vintage year, but also some consistency. For example, all 4 barrels of the 2020 vintage sampled had a much broader diversity of bacteria than all of the other vintages, and this was consistent between the 4 barrels sampled. The dominant fungal microbes were much more varied in this study. The 2016 vintage had a large proportion of ''Penicillium'' (74.8%). Others detected include ''Saccharomyces'' (4.67%), ''Aspergillus'' (4.55%), ''Hanseniaspora'' (4.33%) and ''Brettanomyces'' (4.5%). ''Cladosporium'', ''Rhodotorula'' and ''Torulaspora'' were found in small amounts (all making up less than 2%). The most abundant fungi in the 2017 vintage were ''Brettanomyces'' (31%). This was followed by ''Aspergillus'' (17.1%), ''Saccharomyces'' (12.8%), ''Debaryomyces'' (11.4%) and ''Penicillium'' (10.7%). Genera unique to this vintage include ''Naganishia'' (1.7%) and ''Pichia'' (0.16%). ''Aspergillus'', the filamentous fungi, was seen to be dominant (44%) in the 2019 vintage. ''Saccharomyces'' (26.9%), ''Penicillium'' (9.34%), ''Hanseniaspora'' (8.8%) ''Brettanomyces'' (5.4%) and ''Cladosporium'' (1.9%) were also reported. There was also a wide variation between barrels sampled for the same vintage. While the climate and proximity to the Pacific ocean of Oamaru, New Zealand might have had some impact on the deviation of microbe populations at this brewery versus Belgian lambic breweries, the authors of the study hypothesized that the much longer history of Belgian lambic breweries where microbes have had a much longer time to adapt and become well established versus the relative youth of the brewery in New Zealand (founded in 2014) might be the cause of this deviation <ref name="Ohwofasa_2025">[https://www.mdpi.com/2076-2607/13/2/224 Ohwofasa, A.; Dhami, M.; Winefield, C.; On, S.L.W. Analysis of Bacterial and Fungal Communities and Organic Acid Content in New Zealand Lambic-Style Beers: A Climatic and Global Perspective. Microorganisms 2025, 13, 224. https://doi.org/10.3390/microorganisms13020224.]</ref>. Worth noting is that many of the species reported in this study are obligate aerobes (they require oxygen) and/or are not known to survive in fermented beverages; therefore, the methodologies of this study have been put into question and studies replicating this one would be ideal before accepting these results <ref>[https://www.facebook.com/groups/MilkTheFunk/posts/9931707053524086/?comment_id=9947459055282219 Dr. Bryan Heit. Milk The Funk Facebook Group comment. 01/28/2025.]</ref>.
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.
See also:
* [https://beerandbrewing.com/podcast-episode-364-thomas-vandelanotte-and-bourgogne-de-flanders/ Craft Beer & Brewing Podcast Episode 364: Lambic is Science, Not Sorcery, for Thomas Vandelanotte of Timmermans.]
* [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.]
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"/>