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→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 samples 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>[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>.